Title:

Methods of making nanotechnological and macromolecular biomimetic structures

United States Patent Application 20080096253

Kind Code:

Abstract:

The present invention is in the fields of nanotechology and biomimetics. In particular, the present invention relates to the use of modified ribosomes to produce biomimetic structures. These biomimetic structures, also known as directed element polymers, are not produced by traditional industrial means but instead are produced by living systems comprising modified ribosomes.

Inventors:

Sunguroff, Alexander (Duxbury, MA, US)

Application Number:

11/636723

Publication Date:

04/24/2008

Filing Date:

12/11/2006

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Primary Class:

530/350

Other Classes:

435/195, 536/24.100, 435/325, 435/70.100

International Classes:

C12P21/00; C07H21/02; C07K14/00; C12N5/06; C12N9/14

Attorney, Agent or Firm:

STERNE, KESSLER, GOLDSTEIN & FOX P.L.L.C. (1100 NEW YORK AVENUE, N.W., WASHINGTON, DC, 20005, US)

Claims:

1. A method of producing a nanotechnological or biomimetic structure comprising: (a) forming a mixture comprising: (i) a modified ribosome, (ii) natural or unnatural coding material, (iii) natural or unnatural substrate molecules for assembly into a nanotechnological or biomimetic structure, and (iv) natural or unnatural factors required for synthesis of the nanotechnological or biomimetic structure during the initiation, elongation, or termination phases of assembly, and (b) reacting the mixture under conditions capable of producing a nanotechnological or biomimetic structure, wherein a nanotechnological or biomimetic structure is produced.

2. The method of claim 1, further comprising the step of: (c) isolating the nanotechnological or biomimetic structure.

3. 3-5. (canceled)

6. The method of claim 1, wherein the substrate of the mixture comprises natural or unnatural amino acids, modified natural or unnatural amino acids, non-amino acid molecules suitable for assembly into a nanotechnological or biomimetic structure, or combinations thereof.

7. 7-9. (canceled)

10. The method of claim 1, wherein the modified ribosome of the mixture is capable of acting as an acceptor molecule for assembling the substrates of the mixture into a nanotechnological or biomimetic structure.

11. (canceled)

12. The method of claim 1, wherein the reacting the mixture in step (b) comprises an in vivo, in vitro, or a cell-free system.

13. The method of claim 1, wherein the modified ribosome, natural or unnatural coding material, natural or unnatural factors of the mixture, and combinations thereof are introduced into the mixture using a genetic delivery system.

14. (canceled)

15. A modified ribosome capable of assembling a nanotechnological or biomimetic structure.

16. 16-19. (canceled)

20. A dual mode in vivo method of producing a nanotechnological or biomimetic structure in a host cell comprising: (a) forming a mixture comprising: (i) a modified ribosome(s), (ii) a natural ribosome(s), (iii) natural or unnatural coding material, (iv) natural or unnatural substrate molecules for assembly into a nanotechnological or biomimetic structure, and (v) natural or unnatural factors required for synthesis of the nanotechnological or biomimetic structure during the initiation, elongation, or termination phases of assembly, and (b) reacting the mixture of step (a) under conditions capable of producing a nanotechnological or biomimetic structure, wherein a nanotechnological or biomimetic structure is produced in a host cell.

21. The method of claim 20, further comprising the step of: (c) isolating the nanotechnological or biomimetic structure.

22. The method of claim 20, further comprising the steps of: (c) mutating the parallel dual mode in vivo pathway of the host cell to produce a different nanotechnological or biomimetic structure from the biomimetic or nanotechnological structure produced in a non-mutated host cell.

23. 23-29. (canceled)

30. The method of claim 20, wherein the modified ribosome of the mixture is capable of acting as an acceptor molecule for assembling the substrates of the mixture into a nanotechnological or biomimetic structure.

31. 31-33. (canceled)

34. A method of modifying a ribosome, the method comprising: (a) observing the degradative process used by hydrolyzing enzymes to break a chemical bond; and (b) modifying the active site of a natural ribosome to produce the reverse reaction of the observed degradative process.

35. 35-36. (canceled)

37. An unnatural acceptor molecule comprising a molecule capable of transporting a substrate to a modified ribosome.

38. The unnatural acceptor molecule of claim 37, wherein the unnatural acceptor molecule is an unnatural tRNA.

39. (canceled)

40. A nanotechnological or biomimetic structure produced using the method of claim 1.

41. The biomimetic structure of claim 40, wherein the structure is a polymer or macrocyclic molecule.

42. A nanotechnological or biomimetic structure produced using the method of claim 6.

43. The biomimetic structure of claim 42, wherein the structure is a polymer or macrocyclic molecule.

44. A nonhuman organism comprising a modified ribosome, wherein the nonhuman organism is capable of synthesizing a nanotechnological or biomimetic structure.

45. A host cell comprising a modified ribosome, wherein the host cell is capable of synthesizing a nanotechnological or biomimetic structure.

46. A method of producing a nanotechnological or biomimetic structure in a host cell comprising: (a) forming a mixture comprising: (i) a modified ribosome(s), (ii) a natural ribosome(s), (iii) natural or unnatural coding material, (iv) natural or unnatural substrate molecules for assembly into a nanotechnological or biomimetic structure, and (v) natural or unnatural factors required for synthesis of the nanotechnological or biomimetic structure during the initiation, elongation, or termination phases of assembly, and (b) reacting the mixture of step (a) under conditions capable of producing a nanotechnological or biomimetic structure, (c) isolating the nanotechnological or biomimetic structure by storing, secreting or directly secreting the nanotechnological or biomimetic structure, wherein a nanotechnological or biomimetic structure is produced in a host cell.

47. The method of claim 46, wherein the isolation is performed by temporal or spatial isolation.

48. (canceled)

49. The method of claim 46, further comprising the steps of: (d) mutating the parallel dual mode in vivo pathway of the host cell to produce a different nanotechnological or biomimetic structure from the biomimetic or nanotechnological structure produced in a non-mutated host cell.

50. 50-52. (canceled)

53. The method of claim 46, wherein the substrate of the mixture comprises natural or unnatural amino acids, modified natural or unnatural amino acids, non-amino acid molecules suitable for assembly into a nanotechnological or biomimetic structure, or combinations thereof.

54. 54-56. (canceled)

57. The method of claim 46, wherein the modified ribosome of the mixture is capable of acting as an acceptor molecule for assembling the substrates of the mixture into a nanotechnological or biomimetic structure.

58. 58-63. (canceled)

Description:

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention is in the fields of nanotechnology and biomimetics. In particular, the present invention relates to the use of modified ribosomes to produce biomimetic structures. These biomimetic structures, also known as directed element polymers, are not produced by traditional industrial means but instead are produced by living systems comprising modified ribosomes.

2. Background Art

1. Evolutionary Conservation of Ribosome Structure and Function

In all cells, deoxyribonucleic acid (DNA) records the information required for running the cell and eventually passes this information to subsequent cell generations. Cells extract the information contained within DNA through the processes of transcription and translation. During transcription, DNA is transcribed into messenger RNA (mRNA). During translation, ribosomes translate the mRNA into amino acids and assemble the amino acids into proteins for use in cellular structures and functions.

Ribosomes are present in both prokaryotic and eukaryotic cells and depending on the cell type are free-floating in the cytoplasm, are bound to endoplasmic reticulum, and/or are located within mitochondria and chloroplasts. Ribosomes found in nature are composed of a large and a small subunit. Each subunit is composed of ribosomal nucleic acid (rRNA) and protein. While prokaryotic and eukaryotic ribosomes possess subunits of different size and composition, the two are similar in structure and function. (See, e.g., http://ntri.tamuk.edu/cell/ribosomes.html; Wilson and Nierhaus, Angew, Chem. Int. Ed. 42: 3464-3486 (2003); Fromont-Racine et al., Gene 313: 17-42 (2003).)

A typical translation reaction involves the formation of a complex between a ribosome, a mRNA codon (a unit of 3 nucleotides), a tRNA covalently linked to one of 20 naturally occurring amino acids (an aminoacyl-tRNA), a tRNA covalently linked to the peptide chain being elongated by translation (a peptidyl-tRNA), and associated factors. A particular mRNA codon is recognized by an aminoacyl-tRNA possessing a complementary sequence (an anti-codon). The ribosome executes its task of building of an ordered sequence of polymerized amino acids, i.e. a protein, by positioning each mRNA, aminoacyl-tRNA and peptidyl-tRNA within specific ribosomal sites such that a bond can be catalyzed between an amino acid and the growing peptide chain. (See, e.g., http://ntri.tamuk.edu/cell/ribosomes.html; Rodnina and Wintermeyer, Curr. Opin. Struct. Biol. 13:334-340 (2003); Wilson and Nierhaus, Angew, Chem. Int. Ed. 42: 3464-3486 (2003); Agris, Nucleic Acids Res. 32: 223-238 (2004); Youngman et al., Cell 117: 589-599 (2004).)

The ribosome is so central to the survival of all living cells that over evolutionary timeframes ribosomes and associated tRNA molecules have been strongly conserved. This is because any radical change to these mechanisms is too traumatic to the cell and results in cell death. It appears that only through human-mediated engineering can the ribosome/tRNA machinery leap over the dead-zones of non-viable states to achieve functioning systems that are radically different from the highly conserved mechanisms of naturally evolved life. Creating such in vitro systems that produce biomimetic products as a result of the engineering is described herein.

2. Modified Ribosomes

Much information has been gained in recent decades regarding the formation of ribosomes and the mechanisms and interactions underlying their structure and function. Medline database searches for terms such as “mutant ribosome” or “altered ribosome” demonstrate that specific changes in ribosome structure and function have been described in the art. Such altered ribosomes include ribosomes with altered mRNA binding affinities (Prescott and Göringer, Nucleic Acids Res. 18: 5381-5386 (1990)), altered co-factor binding affinities (Prescott and Göringer, Nucleic Acids Res. 18: 5381-5386 (1990)), altered aminoacyl-tRNA binding affinities (Braverman et al., Nucleic Acids Res. 2: 501-507 (1975)), altered peptidyl-tRNA binding affinities (Meskauskas and Dinman, RNA 7: 1084-1096 (2001)), altered peptide release (Youngman et al., Cell 117: 589-599 (2004)), altered sensitivity to protein synthesis inhibitors (Ono et al., Mol. Cell. Biol. 2: 599-606 (1982)), altered mRNA:tRNA translocation (Southworth et al., J. Mol. Biol. 324: 611-623 (2002)), and altered peptidyltransferase activities (Thompson et al., Proc. Natl. Acad. Sci. 98: 9002-9007 (2001)) as well as ribosomes that read through nonsense (Prescott and Göringer, Nucleic Acids Res. 18: 5381-5386 (1990)) or stop codons (Thompson et al., Proc. Natl. Acad. Sci. 98: 9002-9007 (2001)), ribosomes that recognize mutated mRNA species (Hui and de Boer, Proc. Natl. Acad. Sci. 84: 4762-4766 (1987)), and ribosomes that allow incorporation of nonproteinogenic amino acids into proteins (Dedkova et al., J. Am. Chem. Soc. 125: 6616-6617 (2003)).

Over thirty years ago, it was shown that the natural ribosome has catalytic activity for forming ester as well as peptide (amide) bonds (Fahnesock and Rich Science 173:340-343 (1971)). Non-ribosomal biological polypeptide synthesis also plays with this amide/ester similarity in its normal cellular operation. Therefore, in order the construct alternative or unnatural biomimetic structures using cellular machinery, the ribosome is an integral part of this alternative biosynthetic pathway.

Recent articles have expanded on what was previously known of the structure of the ribosome. In prokaryotes, much research has been devoted to both the smaller unit, known by its centrifugal weight as the 30S unit, and to the larger subunit, known as the 50S unit. Together the combined intact ribosome is known by its weight as 70S.

In particular the work of Nissen et al. ( Science 289: 920-930 (2000)) and Schluenzen et al. ( Cell , Vol. 102, 615-623, (2000)), both of which are entirely incorporated by reference, provided new insight on the ribosomal subunits. For the 50S subunit, the Nissen et al indicated that the purpose of the 50S subunit is to “weld” the polypeptide chain together through its catalytic active site. For the smaller 30S subunit, Schluenzen et al article showed the purpose of the 30S subunit is to function as a “reading” unit that ingests the mRNA peptide building instructions and then aligns the tRNA-monomer complexes against the 50S polymerizing site.

Understanding the structure and function of both ribosomal subunits and the peptide synthesis process can be critical to reengineering the natural peptide synthesis system to produce the directed element polymers of the present invention. For example, Nissen et al. observed the chemical symmetry between catalysts that form bonds and the catalysts that break the same bonds. Nissen et al. noted the symmetry between the specific atoms of the active site of the ribosome and very similar individual atoms comprising the active site of a hydrolyzing digestive enzyme such as chymotrypsin that degrades proteins into their constituent amino acids.

3. Biomimetics

Biomimetics is a field in which natural biological structures and functions are mimicked using components or systems not utilized for the same structures and functions by biological organisms. The field embraces computational, mechanical, industrial, and biological applications (Drexler, Proc. Natl. Acad. Sci. 78: 5275-5278 (1981); Cui and Gao, Biotechnol. Prog. 19: 683-692 (2003); Sarikaya et al., Nat. Mater. 2: 577-585 (2003)). Biomimetics is also a hybrid field based on nanotechnology and biotechnology in which products and functions of biological systems can be engineered to interact with inorganic compounds for uses in nanotechnology and biotechnology (See e.g., Sarikaya et al., Nat. Mater. 2: 577-585 (2003)). A benefit of such engineering is that biological systems allow for specific recognition of molecular substrates in catalytic reactions and allow for assembly of structures from a molecular level (Id.).

Biomimetics also encompasses the creation of artificial organisms and new biological systems (i.e., biosystems, also encompassing in vitro or cell-free biological components, products, and functions) (See e.g., Liu and Schultz, Proc. Natl. Acad. Sci. 96: 4780-4785 (1999); Hesman, Sci. News 157: 360 (2000); Gibbs, “Synthetic Life,” Apr. 26, 2004 at http://www.sciam.com). Such organisms and biosystems produce unnatural products or carry out unnatural functions. The term ‘unnatural’ in this context means not naturally utilized or produced in the mimicked biological processes.

Biomimetic forms of translation have previously utilized modified forms of tRNA (Liu and Schultz, Proc. Natl. Acad. Sci. 96: 4780-4785 (1999)), modified nucleobases (See e.g., Kool, Acc. Chem. Res. 35: 936-943 (2002)), and modified codon-anticodon pairing (Hohsaka and Sisido, Curr. Opin. Chem. Biol. 4: 645-652 (2002)). In terms of non-biomimetic structures, mutant ribosomes in the art have been utilized to incorporate D-amino acids into proteins (Dedkova et al., J. Am. Chem. Soc. 125: 6616-6617 (2003)). As noted by Drexler, the molecular machinery associated with protein synthesis and function is a powerful tool by which the complex synthetic strategies of conventional organic chemistry can be side-stepped by site-specific synthetic reactions associated with engineered biological mechanisms ( Proc. Natl. Acad. Sci. 78: 5275-5278 (1981)).

Modified ribosomes as contemplated by the invention allow for the possibility of a broader range and greater specificity of synthesis than available with current biomimetic products and methods. For example, the ability of molecules to mimic tRNA structure and function within specific ribosomal binding sites offers a means by which an extensive range of biomimetic products can be produced (See e.g., Wilson and Nierhaus, Angew, Chem. Int. Ed. 42: 3464-3486 (2003)).

Bioplastics have been created since the 19 ^thcentury with early 20 ^thcentury ventures such as Henry Ford's “Soy Plastic Car.” Much of the current interest is in starch polymers, polysaccharides, proteins, polyhydroxyalkanoates, polylactic acid, and polymers of triglycerides. The rapidly growing polylactate (PLA) biodegradable plastics industry is an example of use of biologically derived monomers. PLA has been jointly developed by Cargill and Dow in a project called NatureWorks® LLC. While this method uses bacteria to convert corn sugar to lactic acid and then produce the monomer substrate lactide, it still requires industrial polymerization techniques to create the polymers NatureWorks® PLA and Ingeo.

The current invention overcomes the problems associated with traditional polymerization processes by providing methods of producing biomimetic structures, such as directed element polymers, using modified ribosomes.

BRIEF SUMMARY OF THE INVENTION

The present invention is directed to a method of producing a nanotechnological or biomimetic structure comprising:

- (a) forming a mixture comprising:
  - (i) a modified ribosome,
  - (ii) natural or unnatural coding material,
  - (iii) natural or unnatural substrate molecules for assembly into a nanotechnological or biomimetic structure, and
  - (iv) natural or unnatural factors required for synthesis of the nanotechnological or biomimetic structure during the initiation, elongation, or termination phases of assembly, and
- (b) reacting the mixture under conditions capable of producing a nanotechnological or biomimetic structure,
  wherein a nanotechnological or biomimetic structure is produced.

The present invention is also directed to a dual mode in vivo method of producing a nanotechnological or biomimetic structure in a host cell comprising:

- (c) forming a mixture comprising:
  - (i) a modified ribosome(s),
  - (ii) a natural ribosome(s),
  - (iii) natural or unnatural coding material,
  - (iv) natural or unnatural substrate molecules for assembly into a nanotechnological or biomimetic structure, and
  - (v) natural or unnatural factors required for synthesis of the nanotechnological or biomimetic structure during the initiation, elongation, or termination phases of assembly, and
- (d) reacting the mixture of step (a) under conditions capable of producing a nanotechnological or biomimetic structure,
  wherein a nanotechnological or biomimetic structure is produced in a host cell.

The invention is also directed to a pedestal mount in vitro method of producing a nanotechnological or biomimetic structure comprising

- (a) forming a mixture comprising:
  - (i) a modified ribosome possessing a pedestal in the ribosome structure that serves as an acceptor site for a substrate molecule,
  - (ii) natural or unnatural coding material,
  - (iii) natural or unnatural substrate molecules for assembly into a nanotechnological or biomimetic structure, and
  - (iv) natural or unnatural factors required for synthesis of the nanotechnological or biomimetic structure during the initiation, elongation, or termination phases of assembly, and
- (b) reacting said mixture of step (a) under conditions capable of producing a nanotechnological or biomimetic structure,
- wherein a nanotechnological or biomimetic structure is produced in a host cell.

In some embodiments, the method further comprises the step of isolating the nanotechnological or biomimetic structure.

In some embodiments, the method further comprises the step of mutating the host cell to produce a different nanotechnological or biomimetic structure from the biomimetic or nanotechnological structure produced in a non-mutated host cell.

In some embodiments, the structure produced has at least one dimension on a scale of nanometers.

In some embodiments, the conditions of the methods of producing a nanotechnological or biomimetic structure occur under nonphysiological conditions selected from the group consisting of elevated or reduced pressure, elevated or reduced temperature, elevated or reduced pH, and combinations thereof.

In some embodiments, the unnatural coding material of the mixture comprises modified nucleoside bases or non-nucleoside base replacements.

In some embodiments, the substrate of the mixture comprises natural or unnatural amino acids, modified natural or unnatural amino acids, non-amino acid molecules suitable for assembly into a nanotechnological or biomimetic structure, or combinations thereof. In some embodiments, the substrate of the mixture further comprises a metal.

In some embodiments, the natural or unnatural factors of the mixture further comprise an acceptor molecule selected from the group consisting of natural tRNA, unnatural tRNA, an acceptor molecule capable of interacting with the coding material of the mixture to assemble substrate molecules into a nanotechnological or biomimetic structure, and combinations thereof.

In some embodiments, the acceptor molecule of the mixture interacts with coding material of the mixture in sequences greater or less than three bases or base replacements in length.

In some embodiments, the modified ribosome of the mixture is capable of acting as an acceptor molecule for assembling the substrates of the mixture into a nanotechnological or biomimetic structure.

In some embodiments, the natural or unnatural factors of the mixture comprise natural or unnatural catalysts that transfer substrates to acceptor molecules.

In some embodiments, the reacting of the mixture in step (b) comprises an in vivo, in vitro, or a cell-free system.

In some embodiments, the modified ribosome, natural or unnatural coding material, natural or unnatural factors of the mixture, and combinations thereof are introduced into the mixture using a genetic delivery system. In some embodiments, the genetic delivery system is a virus, plasmid, or other coding material and wherein the conditions are appropriate for expression of the coding material.

The present invention is also directed to a modified ribosome capable of assembling a nanotechnological or biomimetic structure. In some embodiments, the ribosome is modified from a natural ribosome.

In some embodiments, the modified ribosome comprises ribosomal subunits in natural or unnatural combinations. In some embodiments, the ribosomal subunits comprise natural or unnatural mixtures.

In some embodiments, the modified ribosome comprises an unnatural molecular size, molecular weight, or combination thereof.

The present invention is also directed to a method of modifying a ribosome, the method comprising:

- (a) observing the degradative process used by hydrolyzing enzymes to break a chemical bond; and
- (b) modifying the active site of a natural ribosome to produce the reverse reaction of the observed degradative process.

In some embodiments, the reverse reaction is designed to synthesize cellulose. In some embodiments, the reverse reaction is designed to synthesize polylactate.

The present invention is also directed to an unnatural acceptor molecule comprising a molecule capable of transporting a substrate to a modified ribosome. In some embodiments, the unnatural acceptor molecule is an unnatural tRNA.

In some embodiments, the codon sequence length exceeds three nucleotides but is less than ten nucleotides in length.

The present invention is also directed to the nanotechnological or biomimetic structure produced using any of the methods of the present invention. In some embodiments, the biomimetic structure a polymer or macrocyclic molecule.

The present invention is also directed to a nonhuman organism comprising a modified ribosome, wherein the nonhuman organism is capable of synthesizing a nanotechnological or biomimetic structure.

The present invention is also directed to a host cell comprising a modified ribosome, wherein the host cell is capable of synthesizing a nanotechnological or biomimetic structure.

The present invention is also directed to a method of producing a nanotechnological or biomimetic structure in a host cell comprising:

- (a) forming a mixture comprising:
  - (i) a modified ribosome(s),
  - (ii) a natural ribosome(s),
  - (iii) natural or unnatural coding material,
  - (iv) natural or unnatural substrate molecules for assembly into a nanotechnological or biomimetic structure, and
  - (v) natural or unnatural factors required for synthesis of the nanotechnological or biomimetic structure during the initiation, elongation, or termination phases of assembly, and
- (b) reacting the mixture of step (a) under conditions capable of producing a nanotechnological or biomimetic structure,
- (c) isolating the nanotechnological or biomimetic structure by storing, secreting or directly secreting the nanotechnological or biomimetic structure,
  wherein a nanotechnological or biomimetic structure is produced in a host cell. In some embodiments, the isolation is performed by temporal or spatial isolation. In further embodiments, the isolation method comprises modifying signal recognition protein (SRP) carriers, modifying chaperone proteins or modifying cellular translocons. In another embodiment, the invention further comprising the steps of mutating the parallel dual mode in vivo pathway of the host cell to produce a different nanotechnological or biomimetic structure from the biomimetic or nanotechnological structure produced in a non-mutated host cell. In some embodiments, the modified ribosomes are attached to a membrane or is free-floating within the cell.

In some embodiments, spatial isolation comprises separation by enclosure in a membraneous structure. In further embodiments, the membraneous structure is a lipid bilayer

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A and B illustrate in a 3-dimensional graph the possibility space for creating the directed element polymers of the present invention (FIG. 1A) and open-ended class of possible polymers (FIG. 1B).

FIG. 2 illustrates the natural process for synthesizing proteins using a natural ribosome.

FIG. 3 illustrates an exemplary method, the dual mode in vivo method, of synthesizing a directed element polymer using the present invention.

FIG. 4 illustrates the Temporal Mode In Vivo (TMIV) isolation method.

FIG. 5 illustrates the Isolated Mode In Vivo (IMUV) isolation method.

FIG. 6 illustrates the Autologous Mode In Vivo (AMIV) synthesis method.

FIG. 7 illustrates the Multiple Mode In Vivo (MMIV) synthesis method.

DETAILED DESCRIPTION OF THE INVENTION

This invention is directed to modifying a natural ribosome to produce new or existing copolymers, each based on a chemical bond other than the peptide bond, but as with natural proteins, each sequenced by design from a template. These copolymers constructed from a set of monomers using a chemical bond other than a peptide bond are called Directed Element Polymers (DEPs). In some embodiments, the DEPs will show all the versatility of use that characterize proteins. However, these polymers are not produced by industrial means. Instead modified living systems produce the polymers using a method referred to as a Biological Industrial Operational Polymer (BIOP) paradigm.

The terms “unnatural”, “alternative” and “modified” polymers are used interchangeably herein.

As described in the sections to follow, the present invention is directed to a novel method of producing directed element polymers using living systems comprising a modified ribosome, novel copolymers known as directed element polymers, and methods of using the same. The section headings below are for organizational purposes only and are not intended to impart any division or meaning to this document unless specified otherwise.

The BIOP Method

This invention is directed to modified ribosomes capable of assembling biomimetic structures. A biomimetic structure as contemplated by the invention is an unnatural polymer or structure other than a polymer consisting solely of naturally occurring amino acids. As used in this application, this unnatural polymer or structure shall also be referred to as a directed element polymer (DEP) which comprises directed elements. Based on this definition, the mixed amino acid polymer produced by a mutant ribosome as described in Dedkova et al. ( J. Am. Chem. Soc. 125: 6616-6617 (2003)) would not be encompassed by the invention. In fact such polymers consisting solely of mixed D and L amino acids are considered proteins rather than biomimetic structures, given that natural proteins may contain D-amino acids as a result of posttranslational modification or nonribosomal synthesis (See Hohsaka and Sisido, Curr. Opin. Chem. Biol. 4: 645-652 (2002); Dedkova et al., J. Am. Chem. Soc. 125: 6616-6617 (2003)).

The term “about” when used in conjunction with a percentage or other numerical amount means plus or minus 10% of that percentage or other numerical amount. For example, the term “about 80%” would encompass 80% plus or minus 8%.

The term “modified” as used herein means that the item being modified has been changed in form or character. For example, a modified ribosome is a natural ribosome which has been changed in form or character, e.g., so that it can synthesis a DEP.

The term “substrate” refers to a substance which is acted upon. For example a substrate can be the substance upon which an enzyme acts.

The term “coding material” refers to any substance which can be used to record the order with which a substrate will be inserted into a directed element polymer and wherein the substance can function as a template for directing synthesis of a directed element polymer. For example, in some embodiments, the coding material is a nucleic acid sequence. The coding material of the present invention may itself serve as the template for the synthesis of the directed element polymer, e.g., mRNA, or it may require transcription before acting as the template for synthesis, e.g., DNA being transcribed into mRNA.

Insertion of Coding Material into a Host Cell

In some embodiments, the BIOP method begins with the insertion of coding material into a host cell. Any insertion method known to one of skill in the art can be used, for example, propagating the coding material in the host cell using a vector. Suitable techniques for the present invention, for example, are disclosed in Molecular Biology of the Gene, 5th ed. (Cold Spring Harbor Laboratory Press 2004) herein incorporated by reference in its entirety.

In some embodiments, the coding material is a nucleic acid sequence. These nucleic acids, either DNA or RNA, can be inserted to provide the template for synthesizing the desired DEP. Similar to traditional cellular function, the nucleic acid will contain the information needed to correctly sequence the directed elements into a polymer. For example, if DNA is used, the mRNA produced from this DNA sequence can be used by the modified ribosome to determine the order in which each substrate, such as a directed element, is inserted into the DEP. If RNA is used, then the inserted nucleic acid can be the template used to order the substrates, such as directed elements, within the DEP.

In some embodiments, the coding material is inserted into a nonhuman organism comprising a modified ribosome. In some embodiments, the nonhuman organism is selected from the group consisting of a virus, plasmid, bacteria, fungi, or nonhuman eukaryotic cell. In some embodiments, the nonhuman organism comprising a modified ribosome is capable of synthesizing a nanotechnological or biomimetic structure.

In some embodiments, the coding material is inserted into a host cell comprising a modified ribosome. In some embodiments, the host cell is E. coli . In some embodiments, the host cell comprising a modified ribosome is capable of synthesizing a nanotechnological or biomimetic structure.

Attachment of a Substrate to a tRNA

Natural tRNAs are charged by the attachment of an amino acid to the 3′ terminal adenosine nucleotide via a high-energy acyl linkage as described in Molecular Biology of the Gene . In contrast, those tRNAs without an attached amino acid are considered uncharged. The later hydrolysis of the acyl linkage results in a large change in free energy. This energy is released when the bond is broken and is used to help drive the formation of peptide bonds which link amino acids to each other in polypeptide chains.

In some embodiments, the present invention uses a high-energy bond to attach a substrate molecule to a tRNA. In some embodiments, the present invention uses a high-energy acyl linkage to attach a substrate molecule to a tRNA. In some embodiments, the subsequent breakage of the high-energy bond attaching the substrate to the tRNA is used to aid in the formation of the bonds between the substrate molecules used to form a directed element polymer. As one of skill in the art can appreciate, any suitable high-energy bond which can link the desired substrate to the tRNA can be used in the present invention. Suitable bonds can be determined by examining the chemical bonding properties of both the substrate molecule and the tRNA, e.g., to determine the appropriate chemical groups on each substrate to be linked and the proper chemical bond used to link the identified chemical groups.

In some embodiments, the present invention uses a low-energy bond or multiple low energy-bonds to attach a substrate molecule to a tRNA. In some embodiments, the subsequent breakage of the low-energy bond attaching the substrate to the tRNA is used to aid in the formation of the bonds between the substrate molecules used to form a directed element polymer. In some embodiments, the subsequent breakage of multiple low-energy bonds attaching the substrate to the tRNA is used to aid in the formation of the bonds between the substrate molecules to form a directed element polymer. As one of skill in the art can appreciate, any suitable low-energy bond, or combination of low-energy bonds, which can link the desired substrate to the tRNA can be used in the present invention. Suitable bonds can be determined by examining the chemical bonding properties of both the substrate molecule and the tRNA, e.g., to determine the appropriate chemical groups on each substrate to be linked and the proper chemical bond used to link the identified chemical groups.

In some embodiments, the present invention uses natural or modified tRNA synthetases to attach a substrate molecule to a tRNA, unnatural tRNA, or other acceptor molecule. In natural peptide synthesis, each of the twenty amino acids is attached to the appropriate tRNA by a single, dedicated tRNA synthetase. A single tRNA synthetase, while limited to attaching only one amino acid, can attach this amino acid to any number of tRNAs, each of which is responsible for the amino acid. The present invention, in some embodiments, uses a modified tRNA synthetase or other molecule capable of mimicking the function of tRNA, to attach the substrate molecule to the modified or unnatural tRNA used in the BIOP method.

Unnatural tRNA

To produce a biomimetic polymer like a directed element polymer in a modified ribosome, an unnatural acceptor molecule, such as a modified tRNA, must bring the desired substrate to the active site of the ribosome. A natural tRNA molecule is covalently linked to a single amino acid. Using the anticodon, which is complementary to the codon in the mRNA representing the amino acid, the tRNA delivers its amino acid to the active site of the natural ribosome for insertion into the elongating polymer at the correct location.

The present invention uses these concepts to engineer a modified tRNA, or other molecule capable of mimicking the function of tRNA, to deliver non-amino acid substrates from the cytosol to the active site of the modified ribosome. These non-amino acid substrates are then incorporated into the elongating directed element polymer at a location determined by the anti-codon, or similar localizing mechanism, on the modified tRNA or other molecule capable of mimicking the function of tRNA. It is contemplated that elongation of the claimed biomimetic polymers by modified ribosomes can occur in an ordered sequence that mimics the ordered sequence by which amino acids are assembled during natural translation. For in vivo systems, this modified tRNA or other molecule can be designed so that a natural ribosome can not recognize it.

In some embodiments, the anti-codon on the unnatural tRNA is of an unnatural length. The traditional anti-codon is three nucleotides in length and pairs with a codon of equal length. To provide a successful dual mode in vivo system, it may be necessary to engineer codons of length greater than the natural three nucleotide sequences to prevent interference between the BIOP systems and the natural mechanisms which sustain cellular function.

In some embodiments, the anti-codon of the unnatural tRNA has a nucleotide sequence which exceeds 3 nucleotides in length. In some embodiments, the anti-codon of the unnatural tRNA is between about 3 to about 20 nucleotides in length. In some embodiments, the anti-codon of the unnatural tRNA is between about 3 to about 10 nucleotides in length. In some embodiments, the anti-codon of the unnatural tRNA is between about 3 to about 5 nucleotides in length.

As one of skill in the art will recognize, in the embodiments of the present invention where the anti-codon of the unnatural tRNA is of a length longer than 3 nucleotides, the codon sequence found on the mRNA will be of equal length to preserve the complementarity of the two components. Therefore, in some embodiments, the codon has a nucleotide sequence which exceeds 3 nucleotides in length. In some embodiments, the codon between about 3 to about 20 nucleotides in length. In some embodiments, the codon is between about 3 to about 10 nucleotides in length. In some embodiments, the codon is between about 3 to about 5 nucleotides in length.

Modified Ribosomes

The natural ribosome is the essential manufacturing engine of all living cells. Ribosomes read information and assemble and output manufactured substance. Their input is the sequential instructions from messenger ribonucleic acid (mRNA). Their products are sequenced polymerized strings of amino acids where the amino acid molecules are bonded with a peptide bond. These copolymers are proteins and are an example of a naturally occurring DEP.

In some embodiments, the present invention comprises a modified ribosome produced by modification of a natural ribosome. Such modification may be induced using any method of genetic engineering available to one skilled in the art, including but not limited to, mutagenesis and selective screening techniques.

In some embodiments, the A, E, and P sites of a natural ribosome can be modified to accommodate the binding of the unnatural tRNAs of the present invention. These sites are the natural binding sites for aminoacylated-tRNA (A site), peptidyl-tRNA (P site), and tRNAs which have been released after the growing polypeptide chain has been transferred to the aminoacyl-tRNA (E site). These sites can be altered to accommodate the differences in molecular size or primary, secondary, tertiary, or quaternary chemical structure of both the unnatural tRNA and its substrate when compared to a natural tRNA carrying its substrate, for example, an amino acid.

A natural ribosome may be modified structurally based on the requirements needed to form the desired chemical bond. The ribosomal active site may be modified at either the large or small ribosomal component or at both subunits within the present invention. For example, in prokaryotes the active site modifications may be made on either the 50S or 30S subunit. Similarly, in eukaryotes, the active site modifications may be made on either the 60S or 40S subunit. In particular, modifications can be necessary on the 30S subunit, so that only those mRNA instructions intended for use in the modified parallel synthesis system, i.e. the synthesis system present in an otherwise natural cell which forms DEPs, are used in the parallel synthesis system not the natural system. Further, the 50S active site can be modified to allow for the polymerization of novel monomers.

A natural ribosomal active site, for example, the adenosine base on the 23SrRNA in position 2451 on E. coli , can be modified to synthesize the desired bond in the DEP. Because this base is normally conserved, except for position changes, in most life forms (except a few archaebacteria) it appears to be a critical catalyst for ribosomal synthesis reactions by using one of its nitrogen atoms to transfer protons.

The modified ribosome contemplated by the invention includes any combination of ribosomal subunits whether they exist naturally or not. Such subunits may be comprised of unnatural mixtures of proteins, ribosomal RNAs, and other components and may be of molecular sizes or weights that vary from natural subunits.

In some embodiments, the subunits can be modified to alter the ribosomal tunnel and support structure to accommodate bonds with different stiffness, or the tRNA/substrate alignment mechanism in the ribosome to accommodate alternative tRNA's bringing in alternative substrates. For example, the exit tunnel in a natural ribosome can be altered in molecular size and chemical structure to allow unnatural substrates with different molecular sizes, chemical bonds, or chemical binding affinities to pass through the ribosome as they are inserted into a growing DEP.

In some embodiments, this invention is also directed to modified ribosomes with an active site capable of inserting a directed element into a directed element polymer using any known chemical bond. This active site can be designed to synthesize a specific chemical bond by using the symmetry between the reactions for bond synthesis and bond degradation. For example, researchers have noted that there is symmetry between peptide bond synthesis and the acylation step used by serine proteases to degrade these bonds. (Nissen et al., Science 289:920-930 (2000)).

In some embodiments, this invention is directed to examining known enzymatic reactions, e.g., bond degradative reactions, to design a reaction capable of synthesizing the desired chemical bond between a new directed element and the directed element polymer using a modified ribosome. Using this knowledge about the enzymes, e.g., hydrolyzing enzymes or other degradative enzymes, to gain insight on how to effect changes to the catalytic active site of the ribosome is Reverse Reverse Engineering (RRE).

Life uses enzymes to break up other biological polymers. Conversely, except for proteins being constructed using ribosomes, the other biological polymers are constructed using protein enzymes. Examining these paired mechanisms for construction and destruction of natural polymers can be used to determine the ribosomal active site molecules that are required to form the desired chemical bond in the DEP.

This invention contemplates using all known enzymatic reactions as a model for producing a synthesis reaction. Some specific examples include, but are not limited to, using the reactions of reductases, transferases, hydrolases, lyases, isomerases, ligases, amylases, peptidases, and lipases to engineer a synthesis reaction.

Of particular interest, are the hydrolases, examples of which are contained in Table 1 below. The enzyme number listed below corresponds to the Nomenclature Committee of the International Union of Biochemistry and Molecular Biology (NC-IUBMB) system for numbering enzymes.

TABLE 1


Hydrolases
Enzyme Number
(NC-IUBMB system)	Enzyme Name

EC 3.1	Acting on Ester Bonds
EC 3.1.1	Carboxylic Ester Hydrolases
EC 3.1.1.1	carboxylesterase
EC 3.1.1.2	arylesterase
EC 3.1.1.3	triacylglycerol lipase
EC 3.1.1.4	phospholipase A2
EC 3.1.1.5	lysophospholipase
EC 3.1.1.6	acetylesterase
EC 3.1.1.7	acetylcholinesterase
EC 3.1.1.8	cholinesterase
EC 3.1.1.10	tropinesterase
EC 3.1.1.11	pectinesterase
EC 3.1.1.13	sterol esterase
EC 3.1.1.14	chlorophyllase
EC 3.1.1.15	L-arabinonolactonase
EC 3.1.1.17	gluconolactonase
EC 3.1.1.19	uronolactonase
EC 3.1.1.20	tannase
EC 3.1.1.21	retinyl-palmitate esterase
EC 3.1.1.22	hydroxybutyrate-dimer hydrolase
EC 3.1.1.23	acylglycerol lipase
EC 3.1.1.24	3-oxoadipate enol-lactonase
EC 3.1.1.25	1,4-lactonase
EC 3.1.1.26	galactolipase
EC 3.1.1.27	4-pyridoxolactonase
EC 3.1.1.28	acylcarnitine hydrolase
EC 3.1.1.29	aminoacyl-tRNA hydrolase
EC 3.1.1.30	D-arabinonolactonase
EC 3.1.1.31	6-phosphogluconolactonase
EC 3.1.1.32	phospholipase A1
EC 3.1.1.33	6-acetylglucose deacetylase
EC 3.1.1.34	lipoprotein lipase
EC 3.1.1.35	dihydrocoumarin hydrolase
EC 3.1.1.36	limonin-D-ring-lactonase
EC 3.1.1.37	steroid-lactonase
EC 3.1.1.38	triacetate-lactonase
EC 3.1.1.39	actinomycin lactonase
EC 3.1.1.40	orsellinate-depside hydrolase
EC 3.1.1.41	cephalosporin-C deacetylase
EC 3.1.1.42	chlorogenate hydrolase
EC 3.1.1.43	a-amino-acid esterase
EC 3.1.1.44	4-methyloxaloacetate esterase
EC 3.1.1.45	carboxymethylenebutenolidase
EC 3.1.1.46	deoxylimonate A-ring-lactonase
EC 3.1.1.47	1-alkyl-2-acetylglycerophosphocholine esterase
EC 3.1.1.48	fusarinine-C ornithinesterase
EC 3.1.1.49	sinapine esterase
EC 3.1.1.50	wax-ester hydrolase
EC 3.1.1.51	phorbol-diester hydrolase
EC 3.1.1.52	phosphatidylinositol deacylase
EC 3.1.1.53	sialate O-acetylesterase
EC 3.1.1.54	acetoxybutynylbithiophene deacetylase
EC 3.1.1.55	acetylsalicylate deacetylase
EC 3.1.1.56	methylumbelliferyl-acetate deacetylase
EC 3.1.1.57	2-pyrone-4,6-dicarboxylate lactonase
EC 3.1.1.58	N-acetylgalactosaminoglycan deacetylase
EC 3.1.1.59	juvenile-hormone esterase
EC 3.1.1.60	bis(2-ethylhexyl)phthalate esterase
EC 3.1.1.61	protein-glutamate methylesterase
EC 3.1.1.63	11-cis-retinyl-palmitate hydrolase
EC 3.1.1.64	all-trans-retinyl-palmitate hydrolase
EC 3.1.1.65	L-rhamnono-1,4-lactonase
EC 3.1.1.66	5-(3,4-diacetoxybut-1-ynyl)-2,2′-bithiophene deacetylase
EC 3.1.1.67	fatty-acyl-ethyl-ester synthase
EC 3.1.1.68	xylono-1,4-lactonase
EC 3.1.1.70	cetraxate benzylesterase
EC 3.1.1.71	acetylalkylglycerol acetylhydrolase
EC 3.1.1.72	acetylxylan esterase
EC 3.1.1.73	feruloyl esterase
EC 3.1.1.74	cutinase
EC 3.1.1.75	poly(3-hydroxybutyrate) depolymerase
EC 3.1.1.76	poly(3-hydroxyoctanoate) depolymerase acyloxyacyl hydrolase
EC 3.1.1.77	acyloxyacyl hydrolase
EC 3.1.1.78	polyneuridine-aldehyde esterase
EC 3.1.1.79	hormone-sensitive lipase
EC 3.1.2	Thiolester Hydrolases
EC 3.1.2.1	acetyl-CoA hydrolase
EC 3.1.2.2	palmitoyl-CoA hydrolase
EC 3.1.2.3	succinyl-CoA hydrolase
EC 3.1.2.4	3-hydroxyisobutyryl-CoA hydrolase
EC 3.1.2.5	hydroxymethylglutaryl-CoA hydrolase
EC 3.1.2.6	hydroxyacylglutathione hydrolase
EC 3.1.2.7	glutathione thiolesterase
EC 3.1.2.10	formyl-CoA hydrolase
EC 3.1.2.11	acetoacetyl-CoA hydrolase
EC 3.1.2.12	S-formylglutathione hydrolase
EC 3.1.2.13	S-succinylglutathione hydrolase
EC 3.1.2.14	oleoyl-[acyl-carrier-protein] hydrolase
EC 3.1.2.15	ubiquitin thiolesterase
EC 3.1.2.16	[citrate-(pro-3S)-lyase] thiolesterase
EC 3.1.2.17	(S)-methylmalonyl-CoA hydrolase
EC 3.1.2.18	ADP-dependent short-chain-acyl-CoA hydrolase
EC 3.1.2.19	ADP-dependent medium-chain-acyl-CoA hydrolase
EC 3.1.2.20	acyl-CoA hydrolase
EC 3.1.2.21	dodecanoyl-[acyl-carrier protein] hydrolase
EC 3.1.2.22	palmitoyl[protein] hydrolase
EC 3.1.2.23	4-hydroxybenzoyl-CoA thioesterase
EC 3.1.2.24	2-(2-hydroxyphenyl)benzenesulfinate hydrolase
EC 3.1.2.25	phenylacetyl-CoA hydrolase
EC 3.1.3	Phosphoric Monoester Hydrolases
EC 3.1.3.1	alkaline phosphatase
EC 3.1.3.2	acid phosphatase
EC 3.1.3.3	phosphoserine phosphatase
EC 3.1.3.4	phosphatidate phosphatase
EC 3.1.3.5	5′-nucleotidase
EC 3.1.3.6	3′-nucleotidase
EC 3.1.3.7	3′(2′),5′-bisphosphate nucleotidase
EC 3.1.3.8	3-phytase
EC 3.1.3.9	glucose-6-phosphatase
EC 3.1.3.10	glucose-1-phosphatase
EC 3.1.3.11	fructose-bisphosphatase
EC 3.1.3.12	trehalose-phosphatase
EC 3.1.3.13	bisphosphoglycerate phosphatase
EC 3.1.3.14	methylphosphothioglycerate phosphatase
EC 3.1.3.15	histidinol-phosphatase
EC 3.1.3.16	phosphoprotein phosphatase
EC 3.1.3.17	[phosphorylase] phosphatase
EC 3.1.3.18	phosphoglycolate phosphatase
EC 3.1.3.19	glycerol-2-phosphatase
EC 3.1.3.20	phosphoglycerate phosphatase
EC 3.1.3.21	glycerol-1-phosphatase
EC 3.1.3.22	mannitol-1-phosphatase
EC 3.1.3.23	sugar-phosphatase
EC 3.1.3.24	sucrose-phosphatase
EC 3.1.3.25	inositol-phosphate phosphatase
EC 3.1.3.26	4-phytase
EC 3.1.3.27	phosphatidylglycerophosphatase
EC 3.1.3.28	ADPphosphoglycerate phosphatase
EC 3.1.3.29	N-acylneuraminate-9-phosphatase
EC 3.1.3.31	nucleotidase
EC 3.1.3.32	polynucleotide 3′-phosphatase
EC 3.1.3.33	polynucleotide 5′-phosphatase
EC 3.1.3.34	deoxynucleotide 3′-phosphatase
EC 3.1.3.35	thymidylate 5′-phosphatase
EC 3.1.3.36	phosphoinositide 5-phosphatase
EC 3.1.3.37	sedoheptulose-bisphosphatase
EC 3.1.3.38	3-phosphoglycerate phosphatase
EC 3.1.3.39	streptomycin-6-phosphatase
EC 3.1.3.40	guanidinodeoxy-scyllo-inositol-4-phosphatase
EC 3.1.3.41	4-nitrophenylphosphatase
EC 3.1.3.42	[glycogen-synthase-D] phosphatase
EC 3.1.3.43	[pyruvate dehydrogenase (lipoamide)]-phosphatase
EC 3.1.3.44	[acetyl-CoA carboxylase]-phosphatase
EC 3.1.3.45	3-deoxy-manno-octulosonate-8-phosphatase
EC 3.1.3.46	fructose-2,6-bisphosphate 2-phosphatase
EC 3.1.3.47	[hydroxymethylglutaryl-CoA reductase (NADPH)]-
	phosphatase
EC 3.1.3.48	protein-tyrosine-phosphatase
EC 3.1.3.49	[pyruvate kinase]-phosphatase
EC 3.1.3.50	sorbitol-6-phosphatase
EC 3.1.3.51	dolichyl-phosphatase
EC 3.1.3.52	[3-methyl-2-oxobutanoate dehydrogenase (lipoamide)]-
	phosphatase
EC 3.1.3.53	[myosin-light-chain] phosphatase
EC 3.1.3.54	fructose-2,6-bisphosphate 6-phosphatase
EC 3.1.3.55	caldesmon-phosphatase
EC 3.1.3.56	inositol-polyphosphate 5-phosphatase
EC 3.1.3.57	inositol-1,4-bisphosphate 1-phosphatase
EC 3.1.3.58	sugar-terminal-phosphatase
EC 3.1.3.59	alkylacetylglycerophosphatase
EC 3.1.3.60	phosphoenolpyruvate phosphatase
EC 3.1.3.62	multiple inositol-polyphosphate phosphatase
EC 3.1.3.63	2-carboxy-D-arabinitol-1-phosphatase
EC 3.1.3.64	phosphatidylinositol-3-phosphatase
EC 3.1.3.66	phosphatidylinositol-3,4-bisphosphate 4-phosphatase
EC 3.1.3.67	phosphatidylinositol-3,4,5-trisphosphate 3-phosphatase
EC 3.1.3.68	2-deoxyglucose-6-phosphatase
EC 3.1.3.69	glucosylglycerol 3-phosphatase
EC 3.1.3.70	mannosyl-3-phosphoglycerate phosphatase
EC 3.1.3.71	2-phosphosulfolactate phosphatase
EC 3.1.3.72	5-phytase
EC 3.1.3.73	a-ribazole phosphatase
EC 3.1.3.74	pyridoxal phosphatase
EC 3.1.3.75	phosphoethanolamine/phosphocholine phosphatase
EC 3.1.4	Phosphoric Diester Hydrolases
EC 3.1.4.1	phosphodiesterase I
EC 3.1.4.2	glycerophosphocholine phosphodiesterase
EC 3.1.4.3	phospholipase C
EC 3.1.4.4	phospholipase D
EC 3.1.4.11	phosphoinositide phospholipase C
EC 3.1.4.12	sphingomyelin phosphodiesterase
EC 3.1.4.13	serine-ethanolaminephosphate phosphodiesterase
EC 3.1.4.14	[acyl-carrier-protein] phosphodiesterase
EC 3.1.4.15	adenylyl-[glutamate#%G-#%@ammonia ligase]
	hydrolase
EC 3.1.4.16	2′,3′-cyclic-nucleotide 2′-phosphodiesterase
EC 3.1.4.17	3′,5′-cyclic-nucleotide phosphodiesterase
EC 3.1.4.35	3′,5′-cyclic-GMP phosphodiesterase
EC 3.1.4.37	2′,3′-cyclic-nucleotide 3′-phosphodiesterase
EC 3.1.4.38	glycerophosphocholine cholinephosphodiesterase
EC 3.1.4.39	alkylglycerophosphoethanolamine phosphodiesterase
EC 3.1.4.40	CMP-N-acylneuraminate phosphodiesterase
EC 3.1.4.41	sphingomyelin phosphodiesterase D
EC 3.1.4.42	glycerol-1,2-cyclic-phosphate 2-phosphodiesterase
EC 3.1.4.43	glycerophosphoinositol inositolphosphodiesterase
EC 3.1.4.44	glycerophosphoinositol glycerophosphodiesterase
EC 3.1.4.45	N-acetylglucosamine-1-phosphodiester a-N-
	acetylglucosaminidase
EC 3.1.4.46	glycerophosphodiester phosphodiesterase
EC 3.1.4.48	dolichylphosphate-glucose phosphodiesterase
EC 3.1.4.49	dolichylphosphate-mannose phosphodiesterase
EC 3.1.4.50	glycosylphosphatidylinositol phospholipase D
EC 3.1.4.51	glucose-1-phospho-D-mannosylglycoprotein
	phosphodiesterase
EC 3.1.5	Triphosphoric Monoester Hydrolases
EC 3.1.5.1	dGTPase
EC 3.1.6	Sulfuric Ester Hydrolases
EC 3.1.6.1	arylsulfatase
EC 3.1.6.2	steryl-sulfatase
EC 3.1.6.3	glycosulfatase
EC 3.1.6.4	N-acetylgalactosamine-6-sulfatase
EC 3.1.6.6	choline-sulfatase
EC 3.1.6.7	cellulose-polysulfatase
EC 3.1.6.8	cerebroside-sulfatase
EC 3.1.6.9	chondro-4-sulfatase
EC 3.1.6.10	chondro-6-sulfatase
EC 3.1.6.11	disulfoglucosamine-6-sulfatase
EC 3.1.6.12	N-acetylgalactosamine-4-sulfatase
EC 3.1.6.13	iduronate-2-sulfatase
EC 3.1.6.14	N-acetylglucosamine-6-sulfatase
EC 3.1.6.15	N-sulfoglucosamine-3-sulfatase
EC 3.1.6.16	monomethyl-sulfatase
EC 3.1.6.17	D-lactate-2-sulfatase
EC 3.1.6.18	glucuronate-2-sulfatase
EC 3.1.7	Diphosphoric Monoester Hydrolases
EC 3.1.7.1	prenyl-diphosphatase
EC 3.1.7.2	guanosine-3′,5′-bis(diphosphate) 3′-diphosphatase
EC 3.1.7.3	monoterpenyl-diphosphatase
EC 3.1.8	Phosphoric Triester Hydrolases
EC 3.1.8.1	aryldialkylphosphatase
EC 3.1.8.2	diisopropyl-fluorophosphatase
EC 3.1.11	Exodeoxyribonucleases Producing 5′-Phosphomonoesters
EC 3.1.11.1	exodeoxyribonuclease I
EC 3.1.11.2	exodeoxyribonuclease III
EC 3.1.11.3	exodeoxyribonuclease (lambda-induced)
EC 3.1.11.4	exodeoxyribonuclease (phage SP3-induced)
EC 3.1.11.5	exodeoxyribonuclease V
EC 3.1.11.6	exodeoxyribonuclease VII
EC 3.1.13	Exoribonucleases Producing 5′-Phosphomonoesters
EC 3.1.13.1	exoribonuclease II
EC 3.1.13.2	exoribonuclease H
EC 3.1.13.3	oligonucleotidase
EC 3.1.13.4	poly(A)-specific ribonuclease
EC 3.1.14	Exoribonucleases Producing 3′-Phosphomonoesters
EC 3.1.14.1	yeast ribonuclease
EC 3.1.15	Exonucleases Active with either Ribo- or
	Deoxyribonucleic Acids and Producing 5′-
	Phosphomonoesters
EC 3.1.15.1	venom exonuclease
EC 3.1.16	Exonucleases Active with either Ribo- or
	Deoxyribonucleic Acids and Producing 3′-
	Phosphomonoesters
EC 3.1.16.1	spleen exonuclease
EC 3.1.21	Endodeoxyribonucleases Producing 5′-
	Phosphomonoesters
EC 3.1.21.1	deoxyribonuclease I
EC 3.1.21.2	deoxyribonuclease IV (phage-T4-induced)
EC 3.1.21.3	type I site-specific deoxyribonuclease
EC 3.1.21.4	type II site-specific deoxyribonuclease
EC 3.1.21.5	type III site-specific deoxyribonuclease
EC 3.1.21.6	CC-preferring endodeoxyribonuclease
EC 3.1.21.7	deoxyribonuclease V
EC 3.1.22	Endodeoxyribonucleases Producing 3′-
	Phosphomonoesters
EC 3.1.22.1	deoxyribonuclease II
EC 3.1.22.2	Aspergillus deoxyribonuclease K1
EC 3.1.22.4	crossover junction endodeoxyribonuclease
EC 3.1.22.5	deoxyribonuclease X
EC 3.1.25	Site-Specific Endodeoxyribonucleases Specific for
	Altered Bases
EC 3.1.25.1	deoxyribonuclease (pyrimidine dimer)
EC 3.1.26	Endoribonucleases Producing 5′-Phosphomonoesters
EC 3.1.26.1	Physarum polycephalum ribonuclease
EC 3.1.26.2	ribonuclease alpha
EC 3.1.26.3	ribonuclease III
EC 3.1.26.4	calf thymus ribonuclease H
EC 3.1.26.5	ribonuclease P
EC 3.1.26.6	ribonuclease IV
EC 3.1.26.7	ribonuclease P4
EC 3.1.26.8	ribonuclease M5
EC 3.1.26.9	ribonuclease [poly-(U)-specific]
EC 3.1.26.10	ribonuclease IX
EC 3.1.26.11	tRNase Z
EC 3.1.27	Endoribonucleases Producing 3′-Phosphomonoesters
EC 3.1.27.1	ribonuclease T2
EC 3.1.27.2	Bacillus subtilis ribonuclease
EC 3.1.27.3	ribonuclease T1
EC 3.1.27.4	ribonuclease U2
EC 3.1.27.5	pancreatic ribonuclease
EC 3.1.27.6	Enterobacter ribonuclease
EC 3.1.27.7	ribonuclease F
EC 3.1.27.8	ribonuclease V
EC 3.1.27.9	tRNA-intron endonuclease
EC 3.1.27.10	rRNA endonuclease
EC 3.1.30	Endoribonucleases Active with either Ribo- or
	Deoxyribonucleic Acids and Producing 5′-
	Phosphomonoesters
EC 3.1.30.1	Aspergillus nuclease S1
EC 3.1.30.2	Serratia marcescens nuclease
EC 3.1.31	Endoribonucleases Active with either Ribo- or
	Deoxyribonucleic Acids and Producing 3′-
	Phosphomonoesters
EC 3.1.31.1	micrococcal nuclease
EC 3.2	Glycosylases
EC 3.2.1	Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl
	compounds
EC 3.2.1.1	a-amylase
EC 3.2.1.2	b-amylase
EC 3.2.1.3	glucan 1,4-a-glucosidase
EC 3.2.1.4	cellulase
EC 3.2.1.6	endo-1,3(4)-b-glucanase
EC 3.2.1.7	inulinase
EC 3.2.1.8	endo-1,4-b-xylanase
EC 3.2.1.10	oligo-1,6-glucosidase
EC 3.2.1.11	dextranase
EC 3.2.1.14	chitinase
EC 3.2.1.15	polygalacturonase
EC 3.2.1.17	lysozyme
EC 3.2.1.18	exo-a-sialidase
EC 3.2.1.20	a-glucosidase
EC 3.2.1.21	b-glucosidase
EC 3.2.1.22	a-galactosidase
EC 3.2.1.23	b-galactosidase
EC 3.2.1.24	a-mannosidase
EC 3.2.1.25	b-mannosidase
EC 3.2.1.26	b-fructofuranosidase
EC 3.2.1.28	A,a-trehalase
EC 3.2.1.31	b-glucuronidase
EC 3.2.1.32	xylan endo-1,3-b-xylosidase
EC 3.2.1.33	amylo-1,6-glucosidase
EC 3.2.1.35	hyaluronoglucosaminidase
EC 3.2.1.36	hyaluronoglucuronidase
EC 3.2.1.37	xylan 1,4-b-xylosidase
EC 3.2.1.38	b-D-fucosidase
EC 3.2.1.39	glucan endo-1,3-b-D-glucosidase
EC 3.2.1.40	a-L-rhamnosidase
EC 3.2.1.41	pullulanase
EC 3.2.1.42	GDP-glucosidase
EC 3.2.1.43	b-L-rhamnosidase
EC 3.2.1.44	fucoidanase
EC 3.2.1.45	glucosylceramidase
EC 3.2.1.46	galactosylceramidase
EC 3.2.1.47	galactosylgalactosylglucosylceramidase
EC 3.2.1.48	sucrose a-glucosidase
EC 3.2.1.49	a-N-acetylgalactosaminidase
EC 3.2.1.50	a-N-acetylglucosaminidase
EC 3.2.1.51	a-L-fucosidase
EC 3.2.1.52	b-L-N-acetylhexosaminidase
EC 3.2.1.53	b-N-acetylgalactosaminidase
EC 3.2.1.54	cyclomaltodextrinase
EC 3.2.1.55	a-N-arabinofuranosidase
EC 3.2.1.56	glucuronosyl-disulfoglucosamine glucuronidase
EC 3.2.1.57	isopullulanase
EC 3.2.1.58	glucan 1,3-b-glucosidase
EC 3.2.1.59	glucan endo-1,3-a-glucosidase
EC 3.2.1.60	glucan 1,4-a-maltotetraohydrolase
EC 3.2.1.61	mycodextranase
EC 3.2.1.62	glycosylceramidase
EC 3.2.1.63	1,2-a-L-fucosidase
EC 3.2.1.64	2,6-b-fructan 6-levanbiohydrolase
EC 3.2.1.65	levanase
EC 3.2.1.66	quercitrinase
EC 3.2.1.67	galacturan 1,4-a-galacturonidase
EC 3.2.1.68	isoamylase
EC 3.2.1.70	glucan 1,6-a-glucosidase
EC 3.2.1.71	glucan endo-1,2-b-glucosidase
EC 3.2.1.72	xylan 1,3-b-xylosidase
EC 3.2.1.73	licheninase
EC 3.2.1.74	glucan 1,4-b-glucosidase
EC 3.2.1.75	glucan endo-1,6-b-glucosidase
EC 3.2.1.76	L-iduronidase
EC 3.2.1.77	mannan 1,2-(1,3)-a-mannosidase
EC 3.2.1.78	mannan endo-1,4-b-mannosidase
EC 3.2.1.80	fructan b-fructosidase
EC 3.2.1.81	agarase
EC 3.2.1.82	exo-poly-a-galacturonosidase
EC 3.2.1.83	k-carrageenase
EC 3.2.1.84	glucan 1,3-a-glucosidase
EC 3.2.1.85	6-phospho-b-galactosidase
EC 3.2.1.86	6-phospho-b-glucosidase
EC 3.2.1.87	capsular-polysaccharide endo-1,3-a-galactosidase
EC 3.2.1.88	b-L-arabinosidase
EC 3.2.1.89	arabinogalactan endo-1,4-b-galactosidase
EC 3.2.1.91	cellulose 1,4-b-cellobiosidase
EC 3.2.1.92	peptidoglycan b-N-acetylmuramidase
EC 3.2.1.93	A,a-phosphotrehalase
EC 3.2.1.94	glucan 1,6-a-isomaltosidase
EC 3.2.1.95	dextran 1,6-a-isomaltotriosidase
EC 3.2.1.96	mannosyl-glycoprotein endo-b-N-acetylglucosaminidase
EC 3.2.1.97	glycopeptide a-N-acetylgalactosaminidase
EC 3.2.1.98	glucan 1,4-a-maltohexaosidase
EC 3.2.1.99	arabinan endo-1,5-a-L-arabinosidase
EC 3.2.1.100	mannan 1,4-mannobiosidase
EC 3.2.1.101	mannan endo-1,6-a-mannosidase
EC 3.2.1.102	blood-group-substance endo-1,4-b-galactosidase
EC 3.2.1.103	keratan-sulfate endo-1,4-b-galactosidase
EC 3.2.1.104	steryl-b-glucosidase
EC 3.2.1.105	strictosidine b-glucosidase
EC 3.2.1.106	mannosyl-oligosaccharide glucosidase
EC 3.2.1.107	protein-glucosylgalactosylhydroxylysine glucosidase
EC 3.2.1.108	lactase
EC 3.2.1.109	endogalactosaminidase
EC 3.2.1.110	mucinaminylserine mucinaminidase
EC 3.2.1.111	1,3-a-L-fucosidase
EC 3.2.1.112	2-deoxyglucosidase
EC 3.2.1.113	mannosyl-oligosaccharide 1,2-a-mannosidase
EC 3.2.1.114	mannosyl-oligosaccharide 1,3-1,6-a-mannosidase
EC 3.2.1.115	branched-dextran exo-1,2-a-glucosidase
EC 3.2.1.116	glucan 1,4-a-maltotriohydrolase
EC 3.2.1.117	amygdalin b-glucosidase
EC 3.2.1.118	prunasin b-glucosidase
EC 3.2.1.119	vicianin b-glucosidase
EC 3.2.1.120	oligoxyloglucan b-glycosidase
EC 3.2.1.121	polymannuronate hydrolase
EC 3.2.1.122	maltose-6′-phosphate glucosidase
EC 3.2.1.123	endoglycosylceramidase
EC 3.2.1.124	3-deoxy-2-octulosonidase
EC 3.2.1.125	raucaffricine b-glucosidase
EC 3.2.1.126	coniferin b-glucosidase
EC 3.2.1.127	1,6-a-L-fucosidase
EC 3.2.1.128	glycyrrhizinate b-glucuronidase
EC 3.2.1.129	endo-a-sialidase
EC 3.2.1.130	glycoprotein endo-a-1,2-mannosidase
EC 3.2.1.131	xylan a-1,2-glucuronosidase
EC 3.2.1.132	chitosanase
EC 3.2.1.133	glucan 1,4-a-maltohydrolase
EC 3.2.1.134	difructose-anhydride synthase
EC 3.2.1.135	neopullulanase
EC 3.2.1.136	glucuronoarabinoxylan endo-1,4-b-xylanase
EC 3.2.1.137	mannan exo-1,2-1,6-a-mannosidase
EC 3.2.1.139	a-glucuronidase
EC 3.2.1.140	lacto-N-biosidase
EC 3.2.1.141	4-a-D-{(14)-a-D-glucano}trehalose trehalohydrolase
EC 3.2.1.142	limit dextrinase
EC 3.2.1.143	poly(ADP-ribose) glycohydrolase
EC 3.2.1.144	3-deoxyoctulosonase
EC 3.2.1.145	galactan 1,3-b-galactosidase
EC 3.2.1.146	b-galactofuranosidase
EC 3.2.1.147	thioglucosidase
EC 3.2.1.149	b-primeverosidase
EC 3.2.1.150	oligoxyloglucan reducing-end-specific cellobiohydrolase
EC 3.2.1.151	xyloglucan-specific endo-b-1,4-glucanase
EC 3.2.2	Hydrolysing N-Glycosyl Compounds
EC 3.2.2.1	purine nucleosidase
EC 3.2.2.2	inosine nucleosidase
EC 3.2.2.3	uridine nucleosidase
EC 3.2.2.4	AMP nucleosidase
EC 3.2.2.5	NAD+ nucleosidase
EC 3.2.2.6	NAD(P)+ nucleosidase
EC 3.2.2.7	adenosine nucleosidase
EC 3.2.2.8	ribosylpyrimidine nucleosidase
EC 3.2.2.9	adenosylhomocysteine nucleosidase
EC 3.2.2.10	pyrimidine-5′-nucleotide nucleosidase
EC 3.2.2.11	b-aspartyl-N-acetylglucosaminidase
EC 3.2.2.12	inosinate nucleosidase
EC 3.2.2.13	1-methyladenosine nucleosidase
EC 3.2.2.14	NMN nucleosidase
EC 3.2.2.15	DNA-deoxyinosine glycosylase
EC 3.2.2.16	methylthioadenosine nucleosidase
EC 3.2.2.17	deoxyribodipyrimidine endonucleosidase
EC 3.2.2.19	ADP-ribosylarginine hydrolase
EC 3.2.2.20	DNA-3-methyladenine glycosylase I
EC 3.2.2.21	DNA-3-methyladenine glycosylase II
EC 3.2.2.22	rRNA N-glycosylase
EC 3.2.2.23	DNA-formamidopyrimidine glycosylase
EC 3.2.2.24	ADP-ribosyl-[dinitrogen reductase] hydrolase
EC 3.2.3	Hydrolysing S-Glycosyl Compounds (discontinued)
EC 3.3	Acting on Ether Bonds
EC 3.3.1	Thioether and Trialkylsulfonium Hydrolases
EC 3.3.1.1	adenosylhomocysteinase
EC 3.3.1.2	adenosylmethionine hydrolase
EC 3.3.2	Ether Hydrolases
EC 3.3.2.1	isochorismatase
EC 3.3.2.2	alkenylglycerophosphocholine hydrolase
EC 3.3.2.3	epoxide hydrolase
EC 3.3.2.4	trans-epoxysuccinate hydrolase
EC 3.3.2.5	alkenylglycerophosphoethanolamine hydrolase
EC 3.3.2.6	leukotriene-A4 hydrolase
EC 3.3.2.7	hepoxilin-epoxide hydrolase
EC 3.3.2.8	limonene-1,2-epoxide hydrolase
EC 3.4	Acting on peptide bonds (Peptidases)
EC 3.4.11	Aminopeptidases
EC 3.4.11.1	Leucyl aminopeptidase
EC 3.4.11.2	Membrane alanyl aminopeptidase
EC 3.4.11.3	Cystinyl aminopeptidase
EC 3.4.11.4	Tripeptide aminopeptidase
EC 3.4.11.5	Prolyl aminopeptidase
EC 3.4.11.6	Arginyl aminopeptidase
EC 3.4.11.7	Glutamyl aminopeptidase
EC 3.4.11.9	Xaa-Pro aminopeptidase
EC 3.4.11.10	Bacterial leucyl aminopeptidase
EC 3.4.11.13	Clostridial aminopeptidase
EC 3.4.11.14	Cytosol alanyl aminopeptidase
EC 3.4.11.15	Lysyl aminopeptidase
EC 3.4.11.16	Xaa-Trp aminopeptidase
EC 3.4.11.17	Tryptophanyl aminopeptidase
EC 3.4.11.18	Methionyl aminopeptidase
EC 3.4.11.19	D-Stereospecific aminopeptidase
EC 3.4.11.20	Aminopeptidase Ey
EC 3.4.11.21	aspartyl aminopeptidase
EC 3.4.11.22	Aminopeptidase I
EC 3.4.11.23	PepB aminopeptidase
EC 3.4.13	Dipeptidases
EC 3.4.13.3	Xaa-His dipeptidase
EC 3.4.13.4	Xaa-Arg dipeptidase
EC 3.4.13.5	Xaa-Methyl-His dipeptidase
EC 3.4.13.7	Glu-Glu dipeptidase
EC 3.4.13.9	Xaa-Pro dipeptidase
EC 3.4.13.12	Met-Xaa dipeptidase
EC 3.4.13.17	non-stereospecific dipeptidase
EC 3.4.13.18	cytosol nonspecific dipeptidase
EC 3.4.13.19	membrane dipeptidase
EC 3.4.13.20	b-Ala-His dipeptidase
EC 3.4.13.21	dipeptidase E
EC 3.4.14	Dipeptidyl-peptidases and tripeptidyl-peptidases
EC 3.4.14.1	Dipeptidyl-peptidase I
EC 3.4.14.2	Dipeptidyl-peptidase II
EC 3.4.14.4	Dipeptidyl-peptidase III
EC 3.4.14.5	Dipeptidyl-peptidase IV
EC 3.4.14.6	Dipeptidyl-dipeptidase
EC 3.4.14.9	Tripeptidyl-peptidase I
EC 3.4.14.10	Tripeptidyl-peptidase II
EC 3.4.14.11	Xaa-Pro dipeptidyl-peptidase
EC 3.4.15	Peptidyl-dipeptidases
EC 3.4.15.1	Peptidyl-dipeptidase A
EC 3.4.15.4	Peptidyl-dipeptidase B
EC 3.4.15.5	Peptidyl-dipeptidase Dcp
EC 3.4.16	Serine-type carboxypeptidases
EC 3.4.16.2	Lysosomal Pro-Xaa carboxypeptidase
EC 3.4.16.4	Serine-type D-Ala-D-Ala carboxypeptidase
EC 3.4.16.5	Carboxypeptidase C
EC 3.4.16.6	Carboxypeptidase D
EC 3.4.17	Metallocarboxypeptidases
EC 3.4.17.1	Carboxypeptidase A
EC 3.4.17.2	Carboxypeptidase B
EC 3.4.17.3	Lysine carboxypeptidase
EC 3.4.17.4	Gly-Xaa carboxypeptidase
EC 3.4.17.6	Alanine carboxypeptidase
EC 3.4.17.8	Muramoylpentapeptide carboxypeptidase
EC 3.4.17.10	Carboxypeptidase E
EC 3.4.17.11	Glutamate carboxypeptidase
EC 3.4.17.12	Carboxypeptidase M
EC 3.4.17.13	Muramoyltetrapeptide carboxypeptidase
EC 3.4.17.14	Zinc D-Ala-D-Ala carboxypeptidase
EC 3.4.17.15	Carboxypeptidase A2
EC 3.4.17.16	Membrane Pro-Xaa carboxypeptidase
EC 3.4.17.17	Tubulinyl-Tyr carboxypeptidase
EC 3.4.17.18	Carboxypeptidase T
EC 3.4.17.19	Carboxypeptidase Taq
EC 3.4.17.20	Carboxypeptidase U
EC 3.4.17.21	glutamate carboxypeptidase II
EC 3.4.17.22	Metallocarboxypeptidase D
EC 3.4.18	Cysteine-type carboxypeptidases
EC 3.4.18.1	Cathepsin X
EC 3.4.19	Omega peptidases
EC 3.4.19.1	Acylaminoacyl-peptidase
EC 3.4.19.2	Peptidyl-glycinamidase
EC 3.4.19.3	Pyroglutamyl-peptidase I
EC 3.4.19.5	b-Aspartyl-peptidase
EC 3.4.19.6	Pyroglutamyl-peptidase II
EC 3.4.19.7	N-Formylmethionyl-peptidase
EC 3.4.19.9	g-Glutamyl hydrolase
EC 3.4.19.11	g-D-Glutamyl-meso-diaminopimelate peptidase I
EC 3.4.19.12	ubiquitinyl hydrolase 1
EC 3.4.21	Serine endopeptidases
EC 3.4.21.1	Chymotrypsin
EC 3.4.21.2	Chymotrypsin C
EC 3.4.21.3	Metridin
EC 3.4.21.4	Trypsin
EC 3.4.21.5	Thrombin
EC 3.4.21.6	Coagulation Factor Xa
EC 3.4.21.7	Plasmin
EC 3.4.21.9	Enteropeptidase
EC 3.4.21.10	Acrosin
EC 3.4.21.12	a-Lytic endopeptidase
EC 3.4.21.19	Glutamyl endopeptidase
EC 3.4.21.20	Cathepsin G
EC 3.4.21.21	Coagulation Factor VIIa
EC 3.4.21.22	Coagulation Factor IXa
EC 3.4.21.25	Cucumisin
EC 3.4.21.26	Prolyl oligopeptidase
EC 3.4.21.27	Coagulation Factor XIa
EC 3.4.21.32	Brachyurin
EC 3.4.21.34	Plasma kallikrein
EC 3.4.21.35	Tissue kallikrein
EC 3.4.21.36	Pancreatic elastase
EC 3.4.21.37	Leukocyte elastase
EC 3.4.21.38	Coagulation Factor XIIa
EC 3.4.21.39	Chymase
EC 3.4.21.41	Complement subcomponent C
EC 3.4.21.42	Complement subcomponent C
EC 3.4.21.43	Classical-complement-pathway C3/C5 convertase
EC 3.4.21.45	Complement Factor I
EC 3.4.21.46	Complement Factor D
EC 3.4.21.47	Alternative-complement-pathway C3/C5 convertase
EC 3.4.21.48	Cerevisin
EC 3.4.21.49	Hypodermin C
EC 3.4.21.50	Lysyl endopeptidase
EC 3.4.21.53	Endopeptidase La
EC 3.4.21.54	g-Renin
EC 3.4.21.55	Venombin AB
EC 3.4.21.57	Leucyl endopeptidase
EC 3.4.21.59	Tryptase
EC 3.4.21.60	Scutelarin
EC 3.4.21.61	Kexin
EC 3.4.21.62	Subtilisin
EC 3.4.21.63	Oryzin
EC 3.4.21.64	Endopeptidase K
EC 3.4.21.65	Thermomycolin
EC 3.4.21.66	Thermitase
EC 3.4.21.67	Endopeptidase So
EC 3.4.21.68	t-Plasminogen activator
EC 3.4.21.69	Protein C (activated)
EC 3.4.21.70	Pancreatic endopeptidase E
EC 3.4.21.71	Pancreatic elastase II
EC 3.4.21.72	IgA-specific serine endopeptidase
EC 3.4.21.73	u-Plasminogen activator
EC 3.4.21.74	Venombin A
EC 3.4.21.75	Furin
EC 3.4.21.76	Myeloblastin
EC 3.4.21.77	Semenogelase
EC 3.4.21.78	Granzyme A
EC 3.4.21.79	Granzyme B
EC 3.4.21.80	Streptogrisin A
EC 3.4.21.81	Streptogrisin B
EC 3.4.21.82	Glutamyl endopeptidase II
EC 3.4.21.83	Oligopeptidase B
EC 3.4.21.84	Limulus clotting factor
EC 3.4.21.85	Limulus clotting factor
EC 3.4.21.86	Limulus clotting enzyme
EC 3.4.21.87	Omptin
EC 3.4.21.88	Repressor LexA
EC 3.4.21.89	Signal peptidase I
EC 3.4.21.90	Togavirin
EC 3.4.21.91	Flavivirin
EC 3.4.21.92	Endopeptidase Clp
EC 3.4.21.93	Proprotein convertase 1
EC 3.4.21.94	Proprotein convertase 2
EC 3.4.21.95	Snake venom factor V activator
EC 3.4.21.96	Lactocepin
EC 3.4.21.97	assemblin
EC 3.4.21.98	hepacivirin
EC 3.4.21.99	spermosin
EC 3.4.21.100	pseudomonalisin
EC 3.4.21.101	xanthomonalisin
EC 3.4.21.102	C-terminal processing peptidase
EC 3.4.21.103	physarolisin
EC 3.4.22	Cysteine endopeptidases
EC 3.4.22.1	Cathepsin B
EC 3.4.22.2	papain
EC 3.4.22.3	Ficain
EC 3.4.22.6	Chymopapain
EC 3.4.22.7	Asclepain
EC 3.4.22.8	Clostripain
EC 3.4.22.10	Streptopain
EC 3.4.22.14	Actinidain
EC 3.4.22.15	Cathepsin L
EC 3.4.22.16	Cathepsin H
EC 3.4.22.24	Cathepsin T
EC 3.4.22.25	Glycyl endopeptidase
EC 3.4.22.26	Cancer procoagulant
EC 3.4.22.27	Cathepsin S
EC 3.4.22.28	Picornain 3C
EC 3.4.22.29	Picornain 2A
EC 3.4.22.30	Caricain
EC 3.4.22.31	Ananain
EC 3.4.22.32	Stem bromelain
EC 3.4.22.33	Fruit bromelain
EC 3.4.22.34	legumain
EC 3.4.22.35	Histolysain
EC 3.4.22.36	Caspase-1
EC 3.4.22.37	Gingipain R
EC 3.4.22.38	Cathepsin K
EC 3.4.22.39	adenain
EC 3.4.22.40	bleomycin hydrolase
EC 3.4.22.41	cathepsin F
EC 3.4.22.42	cathepsin O
EC 3.4.22.43	cathepsin V
EC 3.4.22.44	nuclear-inclusion-a endopeptidase
EC 3.4.22.45	helper-component proteinase
EC 3.4.22.46	L-peptidase
EC 3.4.22.47	gingipain K
EC 3.4.22.48	staphopain
EC 3.4.22.49	separase
EC 3.4.22.50	V-cath endopeptidase
EC 3.4.22.51	cruzipain
EC 3.4.22.52	calpain-1
EC 3.4.22.53	calpain-2
EC 3.4.23	Aspartic endopeptidases
EC 3.4.23.1	Pepsin A
EC 3.4.23.2	Pepsin B
EC 3.4.23.3	Gastricsin
EC 3.4.23.4	Chymosin
EC 3.4.23.5	Cathepsin D
EC 3.4.23.12	Nepenthesin
EC 3.4.23.15	Renin
EC 3.4.23.16	HIV-1 retropepsin
EC 3.4.23.17	Pro-opiomelanocortin converting enzyme
EC 3.4.23.18	Aspergillopepsin I
EC 3.4.23.19	Aspergillopepsin II
EC 3.4.23.20	Penicillopepsin
EC 3.4.23.21	Rhizopuspepsin
EC 3.4.23.22	Endothiapepsin
EC 3.4.23.23	Mucorpepsin
EC 3.4.23.24	Candidapepsin
EC 3.4.23.25	Saccharopepsin
EC 3.4.23.26	Rhodotorulapepsin
EC 3.4.23.28	Acrocylindropepsin
EC 3.4.23.29	Polyporopepsin
EC 3.4.23.30	Pycnoporopepsin
EC 3.4.23.31	Scytalidopepsin A
EC 3.4.23.32	Scytalidopepsin B
EC 3.4.23.34	Cathepsin E
EC 3.4.23.35	Barrierpepsin
EC 3.4.23.36	Signal peptidase II
EC 3.4.23.38	Plasmepsin I
EC 3.4.23.39	Plasmepsin II
EC 3.4.23.40	Phytepsin
EC 3.4.23.41	yapsin 1
EC 3.4.23.42	thermopsin
EC 3.4.23.43	prepilin peptidase
EC 3.4.23.44	nodavirus endopeptidase
EC 3.4.23.45	memapsin 1
EC 3.4.23.46	memapsin 2
EC 3.4.23.47	HIV-2 retropepsin
EC 3.4.23.48	plasminogen activator Pla
EC 3.4.24	Metalloendopeptidases
EC 3.4.24.1	Atrolysin A
EC 3.4.24.3	Microbial collagenase
EC 3.4.24.6	Leucolysin
EC 3.4.24.7	Interstitial collagenase
EC 3.4.24.11	Neprilysin
EC 3.4.24.12	Envelysin
EC 3.4.24.13	IgA-specific metalloendopeptidase
EC 3.4.24.14	Procollagen N-endopeptidase
EC 3.4.24.15	Thimet oligopeptidase
EC 3.4.24.16	Neurolysin
EC 3.4.24.17	Stromelysin 1
EC 3.4.24.18	Meprin A
EC 3.4.24.19	Procollagen C-endopeptidase
EC 3.4.24.20	Peptidyl-Lys metalloendopeptidase
EC 3.4.24.21	Astacin
EC 3.4.24.22	Stromelysin 2
EC 3.4.24.23	Matrilysin
EC 3.4.24.24	Gelatinase A
EC 3.4.24.25	Vibriolysin
EC 3.4.24.26	Pseudolysin
EC 3.4.24.27	Thermolysin
EC 3.4.24.28	Bacillolysin
EC 3.4.24.29	Aureolysin
EC 3.4.24.30	Coccolysin
EC 3.4.24.31	Mycolysin
EC 3.4.24.32	b-Lytic metalloendopeptidase
EC 3.4.24.33	Peptidyl-Asp metalloendopeptidase
EC 3.4.24.34	Neutrophil collagenase
EC 3.4.24.35	Gelatinase B
EC 3.4.24.36	Leishmanolysin
EC 3.4.24.37	Saccharolysin
EC 3.4.24.38	gametolysin
EC 3.4.24.39	Deuterolysin
EC 3.4.24.40	Serralysin
EC 3.4.24.41	Atrolysin B
EC 3.4.24.42	Atrolysin C
EC 3.4.24.43	Atroxase
EC 3.4.24.44	Atrolysin E
EC 3.4.24.45	Atrolysin F
EC 3.4.24.46	Adamalysin
EC 3.4.24.47	Horrilysin
EC 3.4.24.48	Ruberlysin
EC 3.4.24.49	Bothropasin
EC 3.4.24.50	Bothrolysin
EC 3.4.24.51	Ophiolysin
EC 3.4.24.52	Trimerelysin I
EC 3.4.24.53	Trimerelysin II
EC 3.4.24.54	Mucrolysin
EC 3.4.24.55	Pitrilysin
EC 3.4.24.56	Insulysin
EC 3.4.24.57	O-Sialoglycoprotein endopeptidase
EC 3.4.24.58	Russellysin
EC 3.4.24.59	Mitochondrial intermediate peptidase
EC 3.4.24.60	Dactylysin
EC 3.4.24.61	Nardilysin
EC 3.4.24.62	Magnolysin
EC 3.4.24.63	Meprin B
EC 3.4.24.64	Mitochondrial processing peptidase
EC 3.4.24.65	Macrophage elastase
EC 3.4.24.66	Choriolysin L
EC 3.4.24.67	Choriolysin H
EC 3.4.24.68	Tentoxilysin
EC 3.4.24.69	Bontoxilysin
EC 3.4.24.70	Oligopeptidase A
EC 3.4.24.71	Endothelin-converting enzyme
EC 3.4.24.72	Fibrolase
EC 3.4.24.73	Jararhagin
EC 3.4.24.74	Fragilysin
EC 3.4.24.75	Lysostaphin
EC 3.4.24.76	flavastacin
EC 3.4.24.77	snapalysin
EC 3.4.24.78	gpr endopeptidase
EC 3.4.24.79	pappalysin-1
EC 3.4.24.80	membrane-type matrix metalloproteinase-1
EC 3.4.24.81	ADAM10 endopeptidase
EC 3.4.24.82	ADAMTS-4 endopeptidase
EC 3.4.24.83	anthrax lethal factor endopeptidase
EC 3.4.24.84	Ste24 endopeptidase
EC 3.4.24.85	S2P endopeptidase
EC 3.4.24.86	ADAM 17 endopeptidase
EC 3.4.25	Threonine endopeptidases
EC 3.4.25.1	proteasome endopeptidase complex
EC 3.5	Acting on Carbon-Nitrogen Bonds, other than Peptide
	Bonds
EC 3.5.1	In Linear Amides
EC 3.5.1.1	asparaginase
EC 3.5.1.2	glutaminase
EC 3.5.1.3	w-amidase
EC 3.5.1.4	amidase
EC 3.5.1.5	urease
EC 3.5.1.6	b-ureidopropionase
EC 3.5.1.7	ureidosuccinase
EC 3.5.1.8	formylaspartate deformylase
EC 3.5.1.9	arylformamidase
EC 3.5.1.10	formyltetrahydrofolate deformylase
EC 3.5.1.11	penicillin amidase
EC 3.5.1.12	biotinidase
EC 3.5.1.13	aryl-acylamidase
EC 3.5.1.14	aminoacylase
EC 3.5.1.15	aspartoacylase
EC 3.5.1.16	acetylornithine deacetylase
EC 3.5.1.17	acyl-lysine deacylase
EC 3.5.1.18	succinyl-diaminopimelate desuccinylase
EC 3.5.1.19	nicotinamidase
EC 3.5.1.20	citrullinase
EC 3.5.1.21	N-acetyl-b-alanine deacetylase
EC 3.5.1.22	pantothenase
EC 3.5.1.23	ceramidase
EC 3.5.1.24	choloylglycine hydrolase
EC 3.5.1.25	N-acetylglucosamine-6-phosphate deacetylase
EC 3.5.1.26	N4-(b-N-acetylglucosaminyl)-L-asparaginase
EC 3.5.1.27	N-formylmethionylaminoacyl-tRNA deformylase
EC 3.5.1.28	N-acetylmuramoyl-L-alanine amidase
EC 3.5.1.29	2-(acetamidomethylene)succinate hydrolase
EC 3.5.1.30	5-aminopentanamidase
EC 3.5.1.31	formylmethionine deformylase
EC 3.5.1.32	hippurate hydrolase
EC 3.5.1.33	N-acetylglucosamine deacetylase
EC 3.5.1.35	D-glutaminase
EC 3.5.1.36	N-methyl-2-oxoglutaramate hydrolase
EC 3.5.1.38	glutamin-(asparagin-)ase
EC 3.5.1.39	alkylamidase
EC 3.5.1.40	acylagmatine amidase
EC 3.5.1.41	chitin deacetylase
EC 3.5.1.42	nicotinamide-nucleotide amidase
EC 3.5.1.43	peptidyl-glutaminase
EC 3.5.1.44	protein-glutamine glutaminase
EC 3.5.1.46	6-aminohexanoate-dimer hydrolase
EC 3.5.1.47	N-acetyldiaminopimelate deacetylase
EC 3.5.1.48	acetylspermidine deacetylase
EC 3.5.1.49	formamidase
EC 3.5.1.50	pentanamidase
EC 3.5.1.51	4-acetamidobutyryl-CoA deacetylase
EC 3.5.1.52	peptide-N4-(N-acetyl-b-glucosaminyl)asparagine amidase
EC 3.5.1.53	N-carbamoylputrescine amidase
EC 3.5.1.54	allophanate hydrolase
EC 3.5.1.55	long-chain-fatty-acyl-glutamate deacylase
EC 3.5.1.56	N,N-dimethylformamidase
EC 3.5.1.57	tryptophanamidase
EC 3.5.1.58	N-benzyloxycarbonylglycine hydrolase
EC 3.5.1.59	N-carbamoylsarcosine amidase
EC 3.5.1.60	N-(long-chain-acyl)ethanolamine deacylase
EC 3.5.1.61	mimosinase
EC 3.5.1.62	acetylputrescine deacetylase
EC 3.5.1.63	4-acetamidobutyrate deacetylase
EC 3.5.1.64	Na-benzyloxycarbonylleucine hydrolase
EC 3.5.1.65	theanine hydrolase
EC 3.5.1.66	2-(hydroxymethyl)-3-(acetamidomethylene)succinate
	hydrolase
EC 3.5.1.67	4-methyleneglutaminase
EC 3.5.1.68	N-formylglutamate deformylase
EC 3.5.1.69	glycosphingolipid deacylase
EC 3.5.1.70	aculeacin-A deacylase
EC 3.5.1.71	N-feruloylglycine deacylase
EC 3.5.1.72	D-benzoylarginine-4-nitroanilide amidase
EC 3.5.1.73	carnitinamidase
EC 3.5.1.74	chenodeoxycholoyltaurine hydrolase
EC 3.5.1.75	urethanase
EC 3.5.1.76	arylalkyl acylamidase
EC 3.5.1.77	N-carbamoyl-D-amino acid hydrolase
EC 3.5.1.78	glutathionylspermidine amidase
EC 3.5.1.79	phthalyl amidase
EC 3.5.1.81	N-acyl-D-amino-acid deacylase
EC 3.5.1.82	N-acyl-D-glutamate deacylase
EC 3.5.1.83	N-acyl-D-aspartate deacylase
EC 3.5.1.84	biuret amidohydrolase
EC 3.5.1.85	(S)—N-acetyl-1-phenylethylamine hydrolase
EC 3.5.1.86	mandelamide amidase
EC 3.5.1.87	N-carbamoyl-L-amino-acid hydrolase
EC 3.5.1.88	peptide deformylase
EC 3.5.1.89	N-acetylglucosaminylphosphatidylinositol deacetylase
EC 3.5.1.90	adenosylcobinamide hydrolase
EC 3.5.2	In Cyclic Amides
EC 3.5.2.1	barbiturase
EC 3.5.2.2	dihydropyrimidinase
EC 3.5.2.3	dihydroorotase
EC 3.5.2.4	carboxymethylhydantoinase
EC 3.5.2.5	allantoinase
EC 3.5.2.6	b-lactamase
EC 3.5.2.7	imidazolonepropionase
EC 3.5.2.9	5-oxoprolinase (ATP-hydrolysing)
EC 3.5.2.10	creatininase
EC 3.5.2.11	L-lysine-lactamase
EC 3.5.2.12	6-aminohexanoate-cyclic-dimer hydrolase
EC 3.5.2.13	2,5-dioxopiperazine hydrolase
EC 3.5.2.14	N-methylhydantoinase (ATP-hydrolysing)
EC 3.5.2.15	cyanuric acid amidohydrolase
EC 3.5.2.16	maleimide hydrolase
EC 3.5.2.17	hydroxyisourate hydrolase
EC 3.5.3	In Linear Amidines
EC 3.5.3.1	arginase
EC 3.5.3.2	guanidinoacetase
EC 3.5.3.3	creatinase
EC 3.5.3.4	allantoicase
EC 3.5.3.5	formiminoaspartate deiminase
EC 3.5.3.6	arginine deiminase
EC 3.5.3.7	guanidinobutyrase
EC 3.5.3.8	formimidoylglutamase
EC 3.5.3.9	allantoate deiminase
EC 3.5.3.10	D-arginase
EC 3.5.3.11	agmatinase
EC 3.5.3.12	agmatine deiminase
EC 3.5.3.13	formiminoglutamate deiminase
EC 3.5.3.14	amidinoaspartase
EC 3.5.3.15	protein-arginine deiminase
EC 3.5.3.16	methylguanidinase
EC 3.5.3.17	guanidinopropionase
EC 3.5.3.18	dimethylargininase
EC 3.5.3.19	ureidoglycolate hydrolase
EC 3.5.3.20	diguanidinobutanase
EC 3.5.3.21	methylenediurea deaminase
EC 3.5.3.22	proclavaminate amidinohydrolase
EC 3.5.4	In Cyclic Amidines
EC 3.5.4.1	cytosine deaminase
EC 3.5.4.2	adenine deaminase
EC 3.5.4.3	guanine deaminase
EC 3.5.4.4	adenosine deaminase
EC 3.5.4.5	cytidine deaminase
EC 3.5.4.6	AMP deaminase
EC 3.5.4.7	ADP deaminase
EC 3.5.4.8	aminoimidazolase
EC 3.5.4.9	methenyltetrahydrofolate cyclohydrolase
EC 3.5.4.10	IMP cyclohydrolase
EC 3.5.4.11	pterin deaminase
EC 3.5.4.12	dCMP deaminase
EC 3.5.4.13	dCTP deaminase
EC 3.5.4.14	deoxycytidine deaminase
EC 3.5.4.15	guanosine deaminase
EC 3.5.4.16	GTP cyclohydrolase I
EC 3.5.4.17	adenosine-phosphate deaminase
EC 3.5.4.18	ATP deaminase
EC 3.5.4.19	phosphoribosyl-AMP cyclohydrolase
EC 3.5.4.20	pyrithiamine deaminase
EC 3.5.4.21	creatinine deaminase
EC 3.5.4.22	1-pyrroline-4-hydroxy-2-carboxylate deaminase
EC 3.5.4.23	blasticidin-S deaminase
EC 3.5.4.24	sepiapterin deaminase
EC 3.5.4.25	GTP cyclohydrolase II
EC 3.5.4.26	diaminohydroxyphosphoribosylaminopyrimidine
	deaminase
EC 3.5.4.27	methenyltetrahydromethanopterin cyclohydrolase
EC 3.5.4.28	S-adenosylhomocysteine deaminase
EC 3.5.4.29	GTP cyclohydrolase IIa
EC 3.5.4.30	dCTP deaminase (dUMP-forming)
EC 3.5.5	In Nitriles
EC 3.5.5.1	nitrilase
EC 3.5.5.2	ricinine nitrilase
EC 3.5.5.4	cyanoalanine nitrilase
EC 3.5.5.5	arylacetonitrilase
EC 3.5.5.6	bromoxynil nitrilase
EC 3.5.5.7	aliphatic nitrilase
EC 3.5.5.8	thiocyanate hydrolase
EC 3.5.99	In Other Compounds
EC 3.5.99.1	riboflavinase
EC 3.5.99.2	thiaminase
EC 3.5.99.3	hydroxydechloroatrazine ethylaminohydrolase
EC 3.5.99.4	N-isopropylammelide isopropylaminohydrolase
EC 3.5.99.5	2-aminomuconate deaminase
EC 3.5.99.6	glucosamine-6-phosphate deaminase
EC 3.5.99.7	1-aminocyclopropane-1-carboxylate deaminase
EC 3.6	Acting on Acid Anhydrides
EC 3.6.1	In Phosphorus-Containing Anhydrides
EC 3.6.1.1	inorganic diphosphatase
EC 3.6.1.2	trimetaphosphatase
EC 3.6.1.3	adenosinetriphosphatase
EC 3.6.1.5	apyrase
EC 3.6.1.6	nucleoside-diphosphatase
EC 3.6.1.7	acylphosphatase
EC 3.6.1.8	ATP diphosphatase
EC 3.6.1.9	nucleotide diphosphatase
EC 3.6.1.10	endopolyphosphatase
EC 3.6.1.11	exopolyphosphatase
EC 3.6.1.12	dCTP diphosphatase
EC 3.6.1.13	ADP-ribose diphosphatase
EC 3.6.1.14	adenosine-tetraphosphatase
EC 3.6.1.15	nucleoside-triphosphatase
EC 3.6.1.16	CDP-glycerol diphosphatase
EC 3.6.1.17	bis(5′-nucleosyl)-tetraphosphatase (asymmetrical)
EC 3.6.1.18	FAD diphosphatase
EC 3.6.1.19	nucleoside-triphosphate diphosphatase
EC 3.6.1.20	5′-acylphosphoadenosine hydrolase
EC 3.6.1.21	ADP-sugar diphosphatase
EC 3.6.1.22	NAD+ diphosphatase
EC 3.6.1.23	dUTP diphosphatase
EC 3.6.1.24	nucleoside phosphoacylhydrolase
EC 3.6.1.25	triphosphatase
EC 3.6.1.26	CDP-diacylglycerol diphosphatase
EC 3.6.1.27	undecaprenyl-diphosphatase
EC 3.6.1.28	thiamine-triphosphatase
EC 3.6.1.29	bis(5′-adenosyl)-triphosphatase
EC 3.6.1.30	m7G(5′)pppN diphosphatase
EC 3.6.1.31	phosphoribosyl-ATP diphosphatase
EC 3.6.1.39	thymidine-triphosphatase
EC 3.6.1.40	guanosine-5′-triphosphate,3′-diphosphate diphosphatase
EC 3.6.1.41	bis(5′-nucleosyl)-tetraphosphatase (symmetrical)
EC 3.6.1.42	guanosine-diphosphatase
EC 3.6.1.43	dolichyldiphosphatase
EC 3.6.1.44	oligosaccharide-diphosphodolichol diphosphatase
EC 3.6.1.45	UDP-sugar diphosphatase
EC 3.6.1.52	diphosphoinositol-polyphosphate diphosphatase
EC 3.6.2	In Sulfonyl-Containing Anhydrides
EC 3.6.2.1	adenylylsulfatase
EC 3.6.2.2	phosphoadenylylsulfatase
EC 3.6.3	Acting on acid anhydrides; catalysing transmembrane
	movement of substances
EC 3.6.3.1	Mg2+-ATPase
EC 3.6.3.2	Mg2+-importing ATPase
EC 3.6.3.3	Cd2+-exporting ATPase
EC 3.6.3.4	Cu2+-exporting ATPase
EC 3.6.3.5	Zn2+-exporting ATPase
EC 3.6.3.6	H+-exporting ATPase
EC 3.6.3.7	Na+-exporting ATPase
EC 3.6.3.8	Ca2+-transporting ATPase
EC 3.6.3.9	Na+/K+-exchanging ATPase
EC 3.6.3.10	H+/K+-exchanging ATPase
EC 3.6.3.11	Cl−-transporting ATPase
EC 3.6.3.12	K+-transporting ATPase
EC 3.6.3.14	H+-transporting two-sector ATPase
EC 3.6.3.15	Na+-transporting two-sector ATPase
EC 3.6.3.16	arsenite-transporting ATPase
EC 3.6.3.17	monosaccharide-transporting ATPase
EC 3.6.3.18	oligosaccharide-transporting ATPase
EC 3.6.3.19	maltose-transporting ATPase
EC 3.6.3.20	glycerol-3-phosphate-transporting ATPase
EC 3.6.3.21	polar-amino-acid-transporting ATPase
EC 3.6.3.22	nonpolar-amino-acid-transporting ATPase
EC 3.6.3.23	oligopeptide-transporting ATPase
EC 3.6.3.24	nickel-transporting ATPase
EC 3.6.3.25	sulfate-transporting ATPase
EC 3.6.3.26	nitrate-transporting ATPase
EC 3.6.3.27	phosphate-transporting ATPase
EC 3.6.3.28	phosphonate-transporting ATPase
EC 3.6.3.29	molybdate-transporting ATPase
EC 3.6.3.30	Fe3+-transporting ATPase
EC 3.6.3.31	polyamine-transporting ATPase
EC 3.6.3.32	quaternary-amine-transporting ATPase
EC 3.6.3.33	vitamin B12-transporting ATPase
EC 3.6.3.34	iron-chelate-transporting ATPase
EC 3.6.3.35	manganese-transporting ATPase
EC 3.6.3.36	taurine-transporting ATPase
EC 3.6.3.37	guanine-transporting ATPase
EC 3.6.3.38	capsular-polysaccharide-transporting ATPase
EC 3.6.3.39	lipopolysaccharide-transporting ATPase
EC 3.6.3.40	teichoic-acid-transporting ATPase
EC 3.6.3.41	heme-transporting ATPase
EC 3.6.3.42	b-glucan-transporting ATPase
EC 3.6.3.43	peptide-transporting ATPase
EC 3.6.3.44	xenobiotic-transporting ATPase
EC 3.6.3.45	steroid-transporting ATPase
EC 3.6.3.46	cadmium-transporting ATPase
EC 3.6.3.47	fatty-acyl-CoA-transporting ATPase
EC 3.6.3.48	a-factor-transporting ATPase
EC 3.6.3.49	channel-conductance-controlling ATPase
EC 3.6.3.50	protein-secreting ATPase
EC 3.6.3.51	mitochondrial protein-transporting ATPase
EC 3.6.3.52	chloroplast protein-transporting ATPase
EC 3.6.3.53	Ag+-exporting ATPase
EC 3.6.4	Acting on acid anhydrides; involved in cellular and
	subcellular movement
EC 3.6.4.1	myosin ATPase
EC 3.6.4.2	dynein ATPase
EC 3.6.4.3	microtubule-severing ATPase
EC 3.6.4.4	plus-end-directed kinesin ATPase
EC 3.6.4.5	minus-end-directed kinesin ATPase
EC 3.6.4.6	vesicle-fusing ATPase
EC 3.6.4.7	peroxisome-assembly ATPase
EC 3.6.4.8	proteasome ATPase
EC 3.6.4.9	chaperonin ATPase
EC 3.6.4.10	non-chaperonin molecular chaperone ATPase
EC 3.6.4.11	nucleoplasmin ATPase
EC 3.6.5	Acting on GTP; involved in cellular and subcellular
	movement
EC 3.6.5.1	heterotrimeric G-protein GTPase
EC 3.6.5.2	small monomeric GTPase
EC 3.6.5.3	protein-synthesizing GTPase
EC 3.6.5.4	signal-recognition-particle GTPase
EC 3.6.5.5	dynamin GTPase
EC 3.6.5.6	tubulin GTPase
EC 3.7	Acting on Carbon-Carbon Bonds
EC 3.7.1	In Ketonic Substances
EC 3.7.1.1	oxaloacetase
EC 3.7.1.2	fumarylacetoacetase
EC 3.7.1.3	kynureninase
EC 3.7.1.4	phloretin hydrolase
EC 3.7.1.5	acylpyruvate hydrolase
EC 3.7.1.6	acetylpyruvate hydrolase
EC 3.7.1.7	b-diketone hydrolase
EC 3.7.1.8	2,6-dioxo-6-phenylhexa-3-enoate hydrolase
EC 3.7.1.9	2-hydroxymuconate-semialdehyde hydrolase
EC 3.7.1.10	cyclohexane-1,3-dione hydrolase
EC 3.8	Acting on Halide Bonds
EC 3.8.1	In C-Halide Compounds
EC 3.8.1.1	alkylhalidase
EC 3.8.1.2	(S)-2-haloacid dehalogenase
EC 3.8.1.3	haloacetate dehalogenase
EC 3.8.1.5	haloalkane dehalogenase
EC 3.8.1.6	4-chlorobenzoate dehalogenase
EC 3.8.1.7	4-chlorobenzoyl-CoA dehalogenase
EC 3.8.1.8	atrazine chlorohydrolase
EC 3.8.1.9	(R)-2-haloacid dehalogenase
EC 3.8.1.10	2-haloacid dehalogenase (configuration-inverting)
EC 3.8.1.11	2-haloacid dehalogenase (configuration-retaining)
EC 3.9	Acting on Phosphorus-Nitrogen Bonds
EC 3.9.1.1	phosphoamidase
EC 3.10	Acting on Sulfur-Nitrogen Bonds
EC 3.10.1.1	N-sulfoglucosamine sulfohydrolase
EC 3.10.1.2	cyclamate sulfohydrolase
EC 3.11	Acting on Carbon-Phosphorus Bonds
EC 3.11.1.1	phosphonoacetaldehyde hydrolase
EC 3.11.1.2	phosphonoacetate hydrolase
EC 3.12	Acting on Sulfur-Sulfur Bonds
EC 3.12.1.1	trithionate hydrolase
EC 3.13	Acting on Carbon-Sulfur Bonds
EC 3.13.1.1	UDP-sulfoquinovose synthase
EC 3.13.1.2	5-deoxyribos-5-ylhomocysteinase

In some embodiments, the changes to the natural ribosome to produce the modified ribosomes of the present invention are derived from the observation that the reverse reaction happens in the serine protease chymotrypsin, where histidine ⁵⁷and serine ¹⁹⁵exchange a proton through the substrate target protein, cleaving it.

A non-limiting example of the method for engineering a modified ribosomal active site of the present invention is to examine enzymes such as the reverse enzyme Kor cellulase or CesA glucosyltransferase to build a ribosomal active site capable of synthesizing cellulose or other polysaccharide.

In Vitro Methods

Running DEP production in vitro requires one to duplicate most of the mechanisms of life in an external environment. Trying to run most of the mechanisms of life in some external “wash” environment can be difficult, but it eliminates the measures which can be required to separate a modified ribosomal production system from the natural synthesis processes of a living cell in a dual mode in vivo system.

In some embodiments, ribosomes can be harvested from cells and attached to endoplasmic reticulum equivalents. Suitable modified tRNAs and mRNA's are then created and mixed with the ribosomes. In some embodiments, this process can be energetically driven using a supply of ATP. Substrate monomers are then mixed with the modified tRNAs. The mixture is then allowed to react and, in some embodiments, the DEPs produced can be later isolated from the reaction mixture. In some embodiments, a pedestal mount in vitro method can be used.

In Vivo Methods

If in vivo methods are used the choice arises to attempt to have modified ribosomes coexist with natural ones within a cell or to supplant them entirely. The present invention is not directed to supplanting all function with the engineered ribosomes because the present invention strives to leave all the protein generating mechanisms alone while simultaneously synthesizing new bonds in a parallel system.

The present invention establishes a parallel system for polymer generation whose machinery does not interfere with or harm, as much as is possible, the normal base protein synthesis machinery of a cell and the rest of the normal base cellular metabolism. Viability of such reengineered cells during polymer synthesis is a preferred but long term viability of the cell is not required. For example, methods such as pulse production where the alternate systems start running, produce DEPs, and then kill the cell, can be used to produce DEPs.

This process of parallel metabolism with minimal modification is referred to as Dual Mode In vivo (DMIV). Once DMIV is established in a cell line, experimentation with the mechanism can be performed with a reduced chance of causing immediate cell death by breaking the normal base metabolism.

Further, explicit programming changes to structures using random variation similar to evolutionary processes, can be used to generate new DMIV products. If the parallel dual mode in vivo synthesis mechanism is non-fatal, then mutation can be used to explore the space of similar synthetic structures. The process of using mutations to explore an engineered space of possibilities while not transgressing on the normal base metabolism coded by the normal base genome is called Induced Parallel Mode Mutation (IPMM).

Directed Element Polymers

The present invention is also directed to biomimetic structures, also known as DEPs. In some embodiments, the present invention is directed to producing DEPs on a nanotech scale.

The term nanotechnological refers to any fabrication technology in which objects are designed and built by the specification and placement of individual atoms or molecules or where at least one dimension is on a scale of nanometers, for example, producing a DEP with a single chemical bond. A nanotechnological structure refers to any structure produced using a nanotechnological process or to any structure that has at least one dimension on a scale of nanometers.

DEPs can be made using a living system comprising a modified ribosome to produce this new class of materials called directed element polymers. These new materials resemble proteins, in that they are structures that have a directed pattern imposed on them. Like proteins, each of these classes of polymers are made from a set of varying monomeric units, i.e. substrate molecules, that are specified by a nucleic acid template. In embodiments of the present invention, where the monomers used to form the polymer are not identical, the polymer will be a copolymer.

Biomimetic structures, also known as DEPs, encompassed by the present invention include any polymer not naturally utilized or produced in the mimicked biological processes. The directed elements may be linked together using chemical bonds and are, as such, not limited to the peptide bonds produced by the natural translational mechanism. For example, this invention is directed to the formation of the following bonds: 1) bonds in natural polymers found in existing life forms, 2) bonds between monomers that are naturally occurring but are not traditionally found in life forms, 3) bonds between monomers that form polymers that are industrially produced, 4) bonds between high-energy monomers, or 5) any other chemical bond capable of linking monomers, either currently known or later discovered.

In some embodiments, the DEPs of the present invention can be any type of copolymer, including, but not limited to block copolymers, statistical copolymers, grafting copolymers, or alternating copolymers.

Bond Selection

Each class of biomimetic structure, i.e., each DEP, is characterized by its polymerization bond. Each of these classes is based on some set of similar monomers that can be bonded together via that bond. Using thousands of bonds and producing millions of different polymeric chains, novel and cost-effective materials and substances can be produced using living systems comprising modified ribosomes that cannot, or currently are not, made by natural life forms or industrial methods. Their physical properties are dependent on the bond used and choice of monomers. In some embodiments, the biomimetic structure produced can be stiff or flexible; long or short; coiled or not coiled; globular; or combinations thereof.

Natural proteins have a multitude of uses because of their geometric properties. For example, because the peptide bond is flexible, the long polymer of amino acids acts like a piece of cooked spaghetti rather than a dry stiff piece. However, various other bonds will have different stiffnesses and resilience, leading to differences in flexibility and durability.

In some embodiments, the present invention is directed to DEPs having stiff bonds. In other embodiments, the present invention is directed to DEPs having flexible bonds.

Bond stiffness in the DEP can be used to determine the proper application for the DEP. For example, stiffer molecules may be better for use as structural or electronic materials, while more flexible ones could be designed to fold in some manner, exposing side groups in ways that enhance catalytic behavior.

In some embodiments, the folding characteristics of the DEP can be used to classify DEPs. As stated earlier, the definition of a protein is a sequenced polymerized string of amino acids. Theoretically being able to predict the folding and self bonding of polymerized amino acids is part of Proteomics. For each set of DEPs produced by the present invention there would be a specific scientific discipline dealing with how that class of molecules bends, folds and rebonds to itself.

Applications for DEPs

Some uses of DEPs produced using the BIOP method are listed below in Table 2. These uses are exemplary and are not intended as limiting as one of skill in the art will recognize a wide variety of other uses for the DEPs produced using this invention. The term “computational” as used in Table 2 means the use of enzyme systems to process chemical signals to have some effect based on a logical computation of these signals, for example, the G protein cascade in cells.

TABLE 2


Applications for DEPs
Type	Purpose

Catalytic	facilitate chemical reactions
Computational	input reactions change of its other properties
Transportational	attaches to other substances
Electrical Insulators	retard electron transport
Electrical Conductors	allow electron transport
Effector	creates movement
Transducer	emit or absorb energy
Structural	forms mechanical structures

Isolation of DEPs

DMIV is an example of a method of isolating artificial subsystems from interacting with natural subsystems. DMIV, along with other methods of locality, can be used to keep the modifications (e.g. DEPs) from upsetting the balance between natural and unnatural polymers. One method to isolate the artificial subsystems is through analogy to the use of computers.

In computer science, many techniques of isolation are used to simplify both the actual coding of the solution as well as abstract meta-level structures describing the solution of the problem. Computer science, as opposed to the physical sciences, is an artificial intellectual construct but has actual code that runs on real computers. In the physical sciences, the machinery or operating code is visualized, but the meta-level description is not comprehended. Code is encapsulated into Subroutines. Variables can be Global if necessary, but are more useful if kept Local to the subroutines. Thus was born the lambda calculus and its association with the class of recursive functions. To further restrict the interaction of code with itself, Operating Systems, Processes, Virtual Machines, Address Spaces, Control Stacks, and Local Environments were invented.

Biological organisms invented membranes to literally encapsulate the reactions it was trying to control and self-perpetuate. While cells appear to be small droplets of ocean of three billion years ago, they are also small tanks limiting the molecules that are interacting to execute the life machine into a small volume across which iterations would probabilistically occur. As evolution has progressed, the pitfalls of universal interaction caused further evolution of localizing structures. Internal membranes such as endoplasmic reticulum and Golgi apparatus led to organelles such as the nucleus. Symbiotic evolution, as described by Margulis, led to the construction of organelles such as the mitochondrion and the chloroplast.

Therefore, there are several ways these natural/unnatural processes can be constructed. For Dual Mode In Vivo (DMIV) (shown in FIG. 3) processes, the processes are set up in parallel to the natural cellular processes, allowing for simultaneous operation. In Temporal Mode In Vivo (TMIV) (shown in FIG. 4), the alternate processes are set up in parallel to the natural processes; however, the processes are functioning at a different time. Lastly, Isolated Mode In Vivo (IMIV) (shown in FIG. 5) allows for the alternate processes to be set up in parallel to the natural processes, however, they occur in membrane isolated spaces. The interactions can be logically isolated with tagged and coded unique interacting regions. To accomplish this, the existing organelle structures are modified that have created partially isolated regions of the cell While these organelles originally formed by symbiosis, their purpose is keep certain reactions isolated from the cytosol.

DMIV uses a modified small ribosomal component to read specified mRNA strings that have an alternative leader sequence, in order to attach to the small ribosomal component. In Autologous Mode In Vivo (AMIV) (shown in FIG. 6), the in vivo process are set up in parallel to natural process that do not use mRNA for input, but specifies tRNA's by finite state machine states of its internal structure.

DMIV can also be generalized to allow more than one artificial pathway. In Multiple Mode In Vivo (MMIV) (shown in FIG. 7), multiple in vivo processes are set up in parallel to natural processes, allowing simultaneous operation. By having multiple simultaneous production systems, structures are made as combinations of different polymers. Modified translocons are reengineered to cluster and attract multiple ribosomes to produce simultaneous polymer chains that interact into larger structures.

In one embodiment of the invention, the isolation of reengineered biomimetic structures from natural ribosomal mechanisms is performed by DMIV. In DMIV, the DNA source code for both reactive elements and target polymers apply to both the natural and alternative pathways, but all further steps of the protein synthesis mechanism are duplicated and the copy modified. Each step in the synthesis process has a new analog: new mRNAs with alternative initiator leaders; new tRNAs to bind the alternative monomers; new synthases to catalyze the monomer/tRNA binding; new content sensitive reader on the small ribosomal component to only attach to mRNA; new synthesizing site on the large ribosomal component as well as ribosomal mating points and tunnel work; new DEP segregation methods for storing, secreting or directly secreting DEPs by modifying signal recognition protein (SRP) carriers, chaperon proteins, the SR61 translocon and the various prokaryote membrane translocons such as secDF and their chloroplast analogs TOC and TIC and the mitochondiral analog TIM.

For Dual Mode In Vivo (DMIV), the processes are set up in parallel to the natural cellular processes, allowing for simultaneous operation. In Temporal Mode In Vivo (TMIV), the alternate processes are set up in parallel to the natural processes, however the processes are functioning at different time. Lastly, Isolated Mode In Vivo (IMUV) allows for the alternate processes to be set up in parallel to the natural processes, however, they occur in membrane isolated spaces.

In certain embodiments of the invention, a lipid layer of any size or depth can be created. Some examples of lipid layers include, but are not limited to a lipid monolayer (e.g., POPC liposomes), lipid bilayer (e.g., hydrogel containing lipophilic groups (like alkanes) to anchor liposomes; Lahiri's amphiphilic anchor lipid surface which binds lipid bilayers (Langmuir 16:7805-7810 (2000)); proteolipid bilayer (attach detergent solublized receptors to surface in an oriented manner and reconstitute lipid membrane around immobilized receptors using lipid micelles while removing detergent ( Analyt. Biochem. 300:132-8 (2000)); use membrane fractions containing over expressed receptors). Lipid surfaces can also be as described in, e.g., Baird et al., Analyt. Biochem. 310:93-99 (2002); Stenlund et al., Analyt. Biochem. 316:243-250 (2003); Abdiche & Myszka, Analyt. Biochem. 328:233-243 (2004); Ferguson et al., Bioconjugate Chem. 16:1457-1483 (2005); U.S. Pat. No. 6,756,078.

New organelle structures can be formed to isolate the interactions, either by alternate structures such as the nucleolus or by using the precepts noted by Margulis and create life within life to isolate the particular structures operating, while using the environment of the surrounding cell to support the operation of the partially isolated mechanism. However, to use these new alternature life structures, limits must be set on the number of cell divisions to limit runaway growth, other multiple growth limiting mechanisms must be used. The most straightforward approach is to require that reengineered organisms need to be supplied with essential growth factors necessary for their survival.

Alternative Input Methods

DMIV uses a modified small ribosomal component to read specified mRNA strings that have an alternative leader sequence, in order to attach to the small ribosomal component. For applications where the DEP is a simple polymer or one with a simple repeating pattern, the reengineered small ribosomal component functioning as an mRNA reader is replaced by a reengineered ribosomal component that has the polymer pattern encoded in its structure. One example of this method is Autologous Mode In Vivo (AMIV), wherein the in vivo synthetic process is set up in parallel to a natural process that does not use mRNA for input but specifies tRNAs by finite state machine states of its internal structure.

DMIV can be generalized to allow for more than one artificial pathway. In Multiple Mode In Vivo (MMIV), multiple in vivo processes are set up in parallel to natural processes, allowing simultaneous operation. By having multiple simultaneous production systems, one is able to make structures that are combinations of different polymers. Modified translocons are reengineered to cluster and attract multiple ribosomes to produce simultaneous polymer chains that interact into larger structures.

Macro Ribosomal Assembly Structures

In one embodiment of the invention, macro ribosomal assembly structures are used for the production of general purpose nanotechnological devices. Particular unfolded and folded DEPs can be used as parts for nanotechnological devices, but proper assembly is necessary for functioning. While new assembly organelles of the complexity of ribosomes and spliceosomes will need to be invented in the future, a first step in that direction is the use of ribosome assemblies using ribosomes operating in conjunction to create structures one level up from single string DEPs.

To achieve the macro synthesis of DEPs, more than one artificial pathway can be constructed in one cell. This generalization of DMIV to produce more than one additional bond based polymer is called Multiple Mode In Vivo production. In MMIV multiple in vivo processes set up in parallel to natural processes, allowing simultaneous operation. By having multiple simultaneous production systems, structures are made that are combinations of different polymers.

In order to generate these combinations, assemblies of ribosomes can be arrayed together to produce simultaneous chains. These arrays can either be in a static relationship with each other or connected in a physically dynamic array. Simple arrays can produce parallel polymer chains that are designed to bond together at specific points. The folding patterns of the individual polymers are changed by the interaction of two or more chains. Dynamic ribosome arrays can be used to produce multiple polymer chain macromolecules that have a physical tension imparted by the energy investment of the dynamic array. Such multiple chain molecules can be called nano-ropes and nano-braids.

Engineered cells can either be run in DMIV or MMIV. In either case, engineered ribosomes can either be free floating, attached to membranes or clustered in parallel groups to generate interacting DEPs. When clustered, the cluster assemblies can also either be free floating or attached to membranes.

In one embodiment of the invention, the modified translocons are reengineered to cluster into larger structures in a membrane. These structures can attract multiple ribosomes to produce simultaneous polymer chains that interact. In another embodiment, free floating ribosomes engage in ribosome-ribosome attachment to produce simultaneous polymer chains that interact. Ribosomes are reengineered to have attachment point proteins incorporated along the equator orthogonal to the polymer tunnel. Ribosomes are then either attached via these points or using larger interstitial materials. For static arrangements of multiple ribosomes, the attachments are relatively simple. For dynamic arrangements use of actin, myosin and components of microtubules, centromeres and flagellar motors are used to ratchet around each other.

Energy Related Methods

The polymers produced by the embodiments of the invention can compete with the petrochemical industry for types of substrates used in the synthesis process. The energy to drive the polymerization process is obtained from biological sources. This can either be through oxidative processes of normal cell metabolism or it can use the photosynthetic abilities of algae and plants to capture solar energy for metabolic use. Once organisms acquire the energy necessary to drive DEP synthesis, excess energy can be stored as produced polymers similar to the investment of energy into production of the polysaccaride cellulose. In fact, many fuels used currently are, in fact, short polymers.

Having generally described this invention, a further understanding can be obtained by reference to the examples provided herein. These examples are for purposes of illustration only and are not intended to be limiting.

EXAMPLES

The present invention can be used to produce either naturally occurring polymers, biomimetic structures, or nanotechnological structures. To use the present invention to produce natural polymers, such as cellulose, the existing natural ribosome system of protein production is examined to identify points which need to be modified to produce the desired polymer. Further, other naturally occurring polymers can be produced using the present invention after an examination of the process for making and degrading the biological polymers such as sugars, starches and other polysaccharides, polylactic acid, polyglycolic acid, fatty acids, polytriglycerides, or polyhydroxyalkanoates.

More complex and alien DEPs are those that use variant monomers with either high energy carbon bonds such acetylene or pure carbon bonds such as graphite or diamond, or use of esoteric elements similar to carbon such as silicon or germanium. The chemical properties of such DEPs are difficult to predict but one of skill in the art could determine them using chemical modeling programs.

In some embodiments, the method of alternate polymer production requires certain changes to be implemented in a host cell, such as E. coli . Importantly, this method leaves the DNA operational mechanisms largely untouched except for the addition of the new programmed coding material required to produce any or all of the following: the nucleic acid template, the modified tRNAs, the modified tRNA synthetases, or the modified ribosomes. Similarly this method leaves the operation of the natural mRNA intact, while creating a novel class of mRNA to feed information to the modified ribosomes. In some embodiments, this separation between natural mRNAs and the ones used in the present invention is accomplished by using a leader recognition induction area that is defined on the ribosome.

After establishing the proper DNA and mRNA templates, a new class of tRNA used to bring the substrate molecules to the modified ribosomes will be created. The attachment of the substrate molecule to the modified tRNA will require, in some embodiments, the creation of a novel tRNA synthetase. The novel tRNA and a tRNA synthetase interact to bring the substrate molecule to the modified ribosome.

The present invention requires modification of a natural ribosome to create the modified ribosomes of the present invention. Such modifications include changing the key catalytic site of the ribosome, for example the A2451 base, to accommodate the formation of a new bond. The body of the ribosome and exit tunnel can be modified structurally to ensure that the generated polymer does not clog the ribosomal exit tunnel. These changes will be implemented by modifying the DNA sequences used to create natural ribosomes and then allowing this DNA to be expressed in a selected host cell.

Within the host cell, the attachment docking areas that attach the ribosome to membranes, such as the endoplasmic reticulum, will have to be modified. Further, the attachment sites on some of the membranes will have to be changed to have cellular mechanisms to process, segregate, excrete, or tolerate the produced polymer. In some embodiments, the DEP will be stored in special vacuoles to prevent injury to the host cell.

Example 1

Peptide Bond (Protein)

The example method described previously can be used to generate novel proteins using the peptide bond in host cells which do not ordinarily synthesize these proteins. Some of the key components for creating a dual mode in vivo system for creating these novel proteins are summarized in Table 3.

TABLE 3


Base Example of Polypeptide
Property	Value

Bond	Peptide
Monomer Set	About 20 (all amino acids)
Codon Length	3
Substrate Viability Compatibility	Total
Active Site Known	Yes
Similar Synthetic Enzymes Known	No
Degradative Enzymes Known	Yes (e.g., chymotrypsin)
Restrictive Leader Class	Type 0 (base, i.e. none)
tRNA Possible for Substrates	Yes
Tunnel Rework Required	No
Product Viability Compatibility	Yes (Exceptions: various natural
	toxins)

Example 2

Cellulose

The following describes a process which can be used to design a method of producing cellulose using the present invention. The designations of first, second, etc. are provided for organizational purposes only. As one of skill in the art will recognize, most steps can be performed in any order to design the production method described below.

First, researchers identify the bond needed to synthesize the DEP desired, for example the glycoside bond used in cellulose. Second, changes needed in the structure and function of the ribosomal active site are identified to produce a similar but reverse direction catalytic mechanism based on examining the reaction of a hydrolyzing enzyme such as Kor cellulase.

Third, for this example, the tRNAs used will incorporate a codon system of 3 nucleotides because this particular DEP does not require linking a vast number of different substrates but instead is composed of simple sugars only, e.g., β-D-Glucose. While a cellulose polymer consisting of only β-D-Glucose is provided as an example, one of skill in the art will recognize that various isomers of cellulose or cellulose-like compounds can be produced by the method of the present invention.

Fourth, tRNA synthetases are designed to attach the simple sugars used as substrate molecules to the modified tRNAs used in the present invention. In some embodiments, following the naming convention of the aminoacyl tRNA synthetases, the synthetases of this embodiment of the present invention can be termed glucoglycosidic synthetases because they will be adding a glucose molecule (instead of an amino acid) to the modified tRNA via a glycosidic bond (instead of an acyl bond). However, any bond can be used to attach the glucose molecules to the modified tRNA not only a glycosidic bond. Fifth, modified tRNAs are designed to carry the simple sugars to the modified ribosome.

Sixth, the reading function of the 30s ribosomal subunit is designed so that the modified ribosome can function parallel to the natural system. As part of designing this parallel synthetic system, mRNA with a unique leader will be designed.

Seventh, the structure of the natural ribosomal tunnel is examined and redesigned so that the DEP, e.g., cellulose, can pass through it without clogging the tunnel.

Eighth, a system for isolating the product, e.g., cellulose, is designed. Such a system can involve isolating the product from the rest of the host cell, particularly useful if the DEP produced is toxic or otherwise harmful to the cell, and then later removing the product or a method of transporting the DEP through the cellular membrane after it has been produced.

Finally, the design changes identified in the preceding steps are translated into a nucleic acid template and inserted into a host cell to begin production of the DEPs.

The example production method of the present invention described previously can be used to generate cellulose in host cells which do not ordinarily synthesize cellulose. Some of the key components for creating a dual mode in vivo system for creating cellulose are summarized in Table 4.

TABLE 4


Example of Cellulose
Property	Value

Bond	Glycosidic
Monomer Set	1 (β-D-glucose)
Codon Length	1 to 20
Substrate Viability Compatibility	Yes
Active Site Known	No
Similar Synthetic Enzymes Known	Yes (CesA glucosyltransferase)
Degradative Enzymes Known	Yes (Kor cellulase)
Restrictive Leader Class	Type I
tRNA Possible for Substrates	Yes
Tunnel Rework Required	Yes
Product Viability Compatibility	Not available

Example 3

Polylactic Acid (PLA)

The example method described previously can be used to generate PLA in host cells which do not ordinarily synthesize PLA. Some of the key components for creating a dual mode in vivo system for creating PLA are summarized in Table 5.

TABLE 5


Example of PLA
	Property	Value

	Bond	Any
	Monomer Set	1
	Codon Length	1 to 20
	Substrate Viability Compatibility	Yes
	Active Site Known	No
	Similar Synthetic Enzymes Known	Yes
	Degradative Enzymes Known	Yes
	Restrictive Leader Class	Type I
	tRNA Possible for Substrates	Yes
	Tunnel Rework Required	Yes
	Product Viability Compatibility	Not available

Example 4

Polyethylene (PE)

The example method described previously can be used to generate PE in host cells which do not ordinarily synthesize PE. Some of the key components for creating a dual mode in vivo system for creating PE are summarized in Table 6.

TABLE 6


Example of PE
	Property	Value

	Bond	Any
	Monomer Set	1
	Codon Length	1 to 20
	Substrate Viability Compatibility	Not available
	Active Site Known	No
	Similar Synthetic Enzymes Known	No
	Degradative Enzymes Known	Yes
	Restrictive Leader Class	Type I
	tRNA Possible for Substrates	Yes
	Tunnel Rework Required	Yes
	Product Viability Compatibility	Not available

Example 5

Polysilane

The example method described previously can be used to generate polysilane in host cells which do not ordinarily synthesize polysilane. Some of the key components for creating a dual mode in vivo system for creating polysilane are summarized in Table 7.

TABLE 7


Example of PS
	Property	Value

	Bond	Any
	Monomer Set	About 5
	Codon Length	1 to 20
	Substrate Viability Compatibility	Not available
	Active Site Known	No
	Similar Synthetic Enzymes Known	No
	Degradative Enzymes Known	Yes
	Restrictive Leader Class	Type I
	tRNA Possible for Substrates	Yes
	Tunnel Rework Required	Yes
	Product Viability Compatibility	Not available

Example 6

Organometallic Polymers

The example method described previously can be used to generate organometallic polymers in host cells which do not ordinarily synthesize organometallic. An organometallic compound is an organic compound comprising a metal. In some embodiments, the metal is directly bound to a carbon atom. Some of the key components for creating a dual mode in vivo system for creating an organometallic polymer are summarized in Table 8.

TABLE 8


Example of Organometallic Compounds
	Property	Value

	Bond	Any
	Monomer Set	About 10
	Codon Length	1 to 20
	Substrate Viability Compatibility	Not available
	Active Site Known	No
	Similar Synthetic Enzymes Known	No
	Degradative Enzymes Known	No
	Restrictive Leader Class	Type I
	tRNA Possible for Substrates	Yes
	Tunnel Rework Required	Yes
	Product Viability Compatibility	Not available

Having now fully described the present invention in some detail by way of illustration for purposes of clarity of understanding, it will be obvious to one of ordinary skill in the art that the same can be performed by modifying or changing the invention within a wide and equivalent range of conditions, formulations and other parameters without affecting the scope of the invention or any specific embodiment thereof, and that such modifications or changes are intended to be encompassed within the scope of the appended claims.

All publications, patents, and patent applications mentioned in this specification are indicative of the level of skill of those skilled in the art to which this invention pertains, and are herein incorporated by reference to the same extent as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated by reference.

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